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Instruction Manual, iQ5 Optical System Software - Bio-Rad

1. Selected wells arehighlighted in yellow black borders Select Deselect modifies wells inall dye layers Fig 6 6 The Select Analyzed Wells for Display Window To select the wells to include in data display 1 In the PCR Quant tab click Display Wells The Select Analyzed Wells for Display window appears Only those wells that are included in the analysis appear in the window Select and de select wells using the same methods described in the analyze wells section on page 51 After you determine which wells to include in analysis click Apply if you want the Select Analyzed Wells for Display window to remain open after the iQ5 software re analyzes the wells If you are satisfied with your selection click OK to close the window and update the wells to include in the analysis If you click Cancel the iQ5 software closes the window and discards all changes The Select Analyzed Wells for Display window displays traces for fluorophores with parameters per the selected fluorophore selector buttons on the amplification chart You can use this dialog box to select all wells for display Restore all traces for display by right clicking on the amplification chart and clicking Show All Traces in the menu 6 3 Standard Curve Chart The standard curve chart appears when for a given fluorophore more than 2 standards with different quantities are defined in the plate setup The fluorophore selector butto
2. Vessel Type Pates gt Soest O meam T Le Fluorophore Probe Primer e hx axe esce E whole Plate loading Select Add Fluorophores oooocoeo Fig 7 7 Creating a Pure Dye Calibration Plate Setup 118 Section 7 Calibrating the Instrument Select Add Fluorophores To choose the fluorophores needed to calibrate the instrument select from the predefined fluorophores or add custom fluorophores by clicking Select Add Fluorophores The Fluor Selector will be displayed Figure 7 8 Wa Bio Rad iQ5 Fluor Selector Add New Fluor Fig 7 8 The Select Add Fluorophores Dialog Box Add a fluorophore from the list displayed by clicking the checkbox next to the fluorophore name To add a custom fluorophore that is not on the list select the appropriate filter pair from the drop down menu Refer to section 9 2 1 for information about system filter specifications and recommended fluorophores Specify the name and color of the custom fluorophore by typing the name in the Fluor Name field and clicking Add New Fluor The new fluorophore name appears in the list for the selected filter pair Click the checkbox next to the new fluorophore name then select the color to specify the fluorophore in the graphic by clicking on the color field that appears Choose contrasting colors for maximum clarity
3. 6 Section 2 Quick Guides 0 eee 7 2 4 Protocol QUICK Guide 1 eicit etes sachet hex tt REP ede cabs Ceu Ero Da cA ERR R PNE YER 7 2 2 Plate Setup Quick Guide 2 re rn ern nennen nnne nnne nnn nn nn nnn nnne 8 2 3 Running a Real Time Experiment Quick Guide eeeseeeeeeeeeeeennm menn 9 2 3 1 Beginning a RU uie eo cease eter enna eea iaa dera uide iaa de epa xua dde Una ERR de RR RUE 9 2 3 2 Monitoring tHE RUD iei ton e Rara aa A H TR RR LDR EMEN aaa 10 2 4 Data Analysis Quick Guides eeeeeeeseeeeeeeee nennen nnne n nennen nnn nnn nnn nnn nnne 10 2 4 1 PCR Quant Tab Quick Guide eeeeeesseeeseeee nennen nennen ener nennen nnns 10 2 4 2 End Point Quick Guide sirrane aaa nennen ener nnn nennen nnn nennen s annes 11 2 4 3 Allelic Discrimination Quick Guide For Multiplex Data Only eere 12 2 4 4 Gene Expression Quick Guide eeeeeee eene nennen nnne nnne nnn nnn nnne 13 2 4 5 Post Run Plate Setup Editing Quick Guide eeeeeeeeeneeeneneennn nme 16 Section 3 Introduction to the iQ5 Optical System Software Version 2 1 18 Section 4 Workshop Module 1 seee ee eee eren nennen nnne n annnm anna ann nnn nnn 19 4 1 Setup Tabs ee eeepc ER Ere eri kar ene eE e cR RF eB cob x rese Roca ok 19 4 1 1 File Browser Section sansi teh dcen cae secete Pa
4. 31 4 3 1 Selecting a Protocol nace eset osten eee eb ek esu LOR E TRA RE DES a RRRLPA ERR RP HR PRECOR DERE ERES ten 31 4 3 2 Editing or Creating a Protocol ccccccceccecccceeecececeeeeeeeeesececesesececesesasaeeseeeeeenenenes 32 4 3 3 Add Protocol Options rcc eiii eee etie nerit anaa aa leere c PRENNE a4 aD 33 7 3 4 Gradient ten ERR rx a DECEDERE RE EE RER EE VE PERRA EFE ERE FERRRRR RR Cre 35 4 3 5 Melt Curve Paaki ioaea netten e ecran els cA ted aeo c ouais dees outer eal odeur eS 36 4 3 6 Sample Protocol Files eeeeeeeeeeeeeeeeeeene nennen nnne nnn nnn nnn nnn nnn nnn nnn 37 4 4 RUM SOU uus n nie ra erecta olore tea ai Crea Deu cana decease Serenade atten 37 4 4 1 Selecting a RUM SQb sai cscs iiie tre raaraa tian LEER XE RERRRRL ERR ER KEIRERRKRL EPA RARE beinara 38 4 4 2 Creating a RUN Set iiie onere HEP e tar EXER RR IEEE e Ra ERR DX RR AE Ray ER EEE AENEA RR E dun 38 4 5 Data File 22222 ie era Pr Lnd ea LER Lec a REDE XE aa RE a LAXE s Lec O aa Pa Pa ca 38 4 5 1 Selecting a Data File rete tere reine enne ane cnra pecu nada rea eu ka Feu ad H aa a Ya gu dpa a Eau 39 4 5 2 OPENING a Data File beer vag te Dou Posee Ee Teo aa Lew eu i eR adora 39 4 5 3 Opening a Pure Dye Calibration file eeeeeeeeeeeeee enne nnne 39 4 5 4 Applying Alternate Pure Dye Calibrations Alternate RME eere 39 4 5 5 Applying Alternate Background or Well F
5. iQ 5 Optical System Software Instruction Manual Compatible with the MyiQ 2 MyiQ and iQ 5 real time PCR detection systems NOTICE TO PURCHASER Bio Rad s real time thermal cyclers are licensed real time thermal cyclers under Applera s United States Patent 6 814 934 B1 for use in research human n vitro diagnostics and for all other fields except the field of veterinary diagnostics Purchase of this instrument conveys a limited non transferable immunity from suit for the purchaser s own internal research and development and for use in applied fields under one or more of U S Patents 5 656 493 5 333 675 5 475 610 claims 1 44 158 160 163 and 167 only and 6 703 236 claims 1 7 only or corresponding claims in their non U S counterparts owned by Applera Corporation No right is conveyed expressly by implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Bio Rad s thermal cyclers and real time thermal cyclers are covered by one or more of the following U S patents or their foreign counterparts owned by Eppendorf AG U S Patent Nos 6 767 512 and 7 074 367 SYBR is a trademark of Molecular Probes Inc Bio Rad Laboratories Inc is licensed by Molecu
6. 4 4 Fig 6 54 Selecting Error Bar options for Gene Expression Analysis Gene Expression Module Context Menu Right clicking on the gene expression chart will reveal the context menu shown in Figure 6 55 N Sorting Option Corrected Std Devs Adjust Graph Restore Graph Copy Graph Print Graph Fig 6 55 The Gene Expression Chart Context Menu Sorting Option Sorting Option Figure 6 56 allows you to sort of the Gene and Condition Names on the chart display When the Sorting Option is selected a Sort Options menu box is displayed The menu box is divided into three sections Groups Options and the Group member list Use the Groups radio buttons to view a list of Gene Names or Condition Names configured in your data set With the Options radio buttons you can elect to organize the names within the Group member list alphabetically or to rearrange these items manually e Alphabetic order The default selection for Groups is Gene Names and the default selection for Options is Alphabetical order The arrows buttons to the right of the group member list can be used to toggle between an alphabetical sort of the group members in ascending or descending order e Manual order To rearrange Gene or Condition Names manually select the radio button for Manual order Select the group member that you would like to re order and use the 94 Section 6 Data Analysis Module arrow buttons to move the member to the
7. Failed to start run The plate setup that you have chosen current seal Film vessel Plates base unit IQ2Emulator and camera IQ2ColorEmulator does not match any of the currently stored Persistent Well Factors calibration files Please review the current calibrations under View gt Calibration Files gt WellFactors and recalibrate your instrument MB Show error details Cause Well factor data not found This error occurs when a run start is attempted using a vessel and seal combination the instrument has not been calibrated for Solution Well Factors vessel and seal type for your plate must match collected Persistent Well Factor calibration files The instrument should ideally be re calibrated for the desired vessel and seal type combination If a run must be started immediately select a vessel and seal type for which the instrument has already been calibrated After the run has completed recalibrate the instrument for the desired vessel and seal combination and back apply this new Well Factor calibration to your data file Refer to section 4 5 5 for further details x D Failed to start run The plate setup that you have chosen current seal Film vessel Plates base unit IQ2Emulator and camera IQ2ColorEmulator does not match any of the currently stored Pure Dye calibration files Please review the current calibrations under View gt Calibration files gt RMEData and recalibrate your instrument
8. 1 In the PCR Quant screen with a opd data file open assess threshold and baseline information for the data file and make changes if necessary Click the Gene Expr tab Make any changes to Gene and Condition for example Sample and Treatment assignments in the gene expression plate interface e Expand the gene expression plate interface view by clicking on the button to make well identification and selection easier Highlight the wells in the gene expression plate interface you wish to edit e Change the gene assignments in these wells by typing your desired name into the gene pull down men then click enter to apply the name to the selected wells e Change the condition assignments in these wells by typing your desired name into the condition pull down menu then click enter to apply the name to the selected wells NOTE The Gene and Condition Names have a character entry limit of 15 characters e Minimize the gene expression plate interface by clicking on the button to return to the standard view of the Gene Expr tab window Click Relative Quantity dCt Click Recalculate to see your results Relative quantity results are graphed The Data Table spreadsheet is accessed by clicking Data Table The Data Table spreadsheet lists the Condition and Gene names and calculated expression values and C values Right click to print or export this data to Excel NOTE Use the Settings tab then click Gene List to enter a
9. 2 Real Time PCR Detection System The MyiQ2 Real Time PCR Detection System is the latest addition to a family of real time PCR detection systems that are comprised of an optical detection module and iCycler thermal cycler The two color MyiQ 2 features a broad spectrum light source tungsten halogen lamp and paired filter optical design to ensure optimal excitation and emission This results in excellent sensitivity and discrimination between multiple fluorophores A CCD detector captures a simultaneous image of all 96 wells of the block resulting in a comprehensive data set illustrating the kinetic behavior of the data during each cycle The iQ 5 Optical System software version 2 1 is used to control and collect and analyze data from the MyiQ2 Real Time PCR Detection System The software is also capable of controlling and collecting and analyzing data from the single color MyiQ and five color iQ 5 Real Time PCR detection systems This manual provides instructions on use of the software applicable to each of these three systems The online Help manual is available at all times by pressing the F1 key 1 2 Setting Up the System Hardware The MyiQ2 MyiQ or iQ5 system should be installed on a clean dry and level surface Identify an appropriate work area for the installation process prior to unpacking any system components The entire installation process should take approximately 15 minutes to complete Handle the optics module and
10. Click Recalculate The End Point Analysis table displays a positive negative or blank label for each unknown under the Unknowns Call column Click Reports to obtain customized reports of the End Point Analysis 2 4 3 Allelic Discrimination Quick Guide For Multiplex Data Only The Allelic Discrimination feature of the Data Analysis module is available post run and offers flexibility for analyzing allelic discrimination data from multiplex PCR experiments You can display samples on a scatter plot based on threshold cycle or relative fluorescence units RFU values at any PCR cycle You can have the iQ5 software automatically make allele calls or you may manually make the allele calls To analyze an allelic discrimination file 1 Within the Workshop module click Data and select your desired data file using the file tree browser Click Analyze The file opens in the PCR Quant tab of the Data Analysis module The iQ5 software automatically chooses the Data Analysis conditions To manually adjust these conditions refer to the PCR Quant Tab You can select or deselect wells that will be included in the analysis by selecting the Analyze Wells checkbox Click the Allelic Disc tab in the Data Analysis module Select the Fluorophores that represent Allele1 and Allele2 in the Assign Fluorophores area 12 Section 2 Quick Guides 7 Choose between the Threshold Cycle and RFU display modes in the Display Mode area to display
11. Fig 6 31 The Automatic and Manual Call Options Automatic Call is the default option Choose Automatic Call for the software to make genotype assignments for every unknown based on the positions of the vertical and horizontal bars and presents those assignments in the data spreadsheet If at least three positive homozygous controls are specified for each fluorophore the positions of the bars are based on the mean and standard deviation of the threshold cycles or RFU values of the controls If insufficient numbers of controls are specified then the position of each bar is determined by the range of threshold cycles or RFU values in the selected fluorophores The positions of the bars may be manually adjusted by dragging them Click Recalculate after any change in the position of the vertical and horizontal bars and the software will make new genotype assignments based on the adjusted positions Choose Manual Call to make the genotype assignments directly into the data spreadsheet or on the plot You can use the manual call feature to change the definitions of one or 72 Section 6 Data Analysis Module more wells on the plot or in the spreadsheet and then return to Automatic Call and the software will position the vertical and horizontal bars based on the new definitions and then make genotype assignments 6 7 5 Manual Calls To make manual calls on the plot 1 Click Manual Call A new box appears in the window Figure 6 32 Al
12. Section 8 User Profiles Section 8 User Profiles This section contains information on the following topics o User Preferences page 122 o User Administration adding and deleting users page 128 o Logging on page 130 o Switching Users page 131 o Changing Password page 131 o Defining Roles page 132 To assist with the management of files and data created by the iQ5 software one or more user profiles may be created for users of the MyiQ2 MyiQ or iQ5 systems User profiles in the iQ5 software consist of e Login name required e Password optional e Permission settings for collecting saving and analyzing data e Preferred defaults user preferences for collecting saving and analyzing data NOTE All user profile information resides on the hard drive of the computer where the software has been installed this means that user profile information cannot be shared over a network When the software is installed the default user profile has the ability to add and configure additional users through the User Profile module The User Profile module is where users can be added their permissions controlled and their preferences set This module of the iQ5 software consists of 2 tabs e User Preferences e Administration 8 1 User Preferences User Preferences found in the User Profile module can be used to customize the preferences of the currently logged in User For each User created in the iQ5 software user specif
13. Selectedfluorophore s not used on plate Remove unused fluorophores T Cause Fluorophores have been selected in the plate setup however no wells on the plate have been edited to contain these fluorophores Solution This is a reminder to check your plate setup Clicking No will return you to the Editing Plate window Clicking Yes will remove the unused fluorophores and save the plate setup 140 Appendices I x Warning Your software is currently running in emulation mode and is not 2 connected to an instrument Proceed No Cause This is not an error message but a warning reminder that the iQ5 software is running in emulation mode In emulation mode the camera is not detecting and collecting data from the plate and is generating emulated run data for demonstration purposes only Solution If you wish the camera to detect and collect data from your experimental plate exit the software From the Start Menu select programs gt Bio Rad gt iQ5 gt iQ5 to restart the software in standard data collection mode 141 Appendices Appendix C Product Ordering Information Catalog Number 170 9790 223 9441 HSS 9601 MSB 1001 TBS 0201 TCS 0803 170 8756 170 9753 170 8791 170 8794 170 8780 Reagents 170 8860 170 8862 170 8880 170 8882 170 8890 170 8891 170 8896 170 8897 170 8892 170 8893 170 8894 170 8895 Product Description MyiQ2 Real Time PCR System includes iCycle
14. If the dataset being analyzed also contains a standard curve the standard curve chart and spreadsheet data will also be included in this report e PCR Quant Graph Only This report template displays only the PCR amplification chart exactly as analyzed and formatted within the PCR Quant module e Std Curve Data Only Landscape This report template includes only the data included in the Standard Curve C Results spreadsheet Although the report on the screen in displayed in the Portrait orientation the Page Layout print settings are pre set to the Landscape orientation Melt Curve Reports e Melt Curve Detailed This report template includes all of the data available in the Melt Curve Peak module of the iQ5 software including the melt curve chart melt peak chart Melt data spreadsheet and analysis parameters In addition if all fluorophores have been selected for display in the PCR Quant module the PCR amplification chart and analysis parameters will also be displayed e Melt Curve Graph Only This report template displays only the graphs available in the Melt Curve Peak module of the iQ5 software such as the melt curve chart and melt peak chart exactly as analyzed and formatted within the Melt Curve Peak module e Melt Curve Only When creating a report from a data set performed as an Melt Curve Only run the Melt Curve Only report option becomes available This report template includes all of the data available in the Melt Cur
15. optical module on a flat surface taking care not to touch the inside of the nose portion of the module 2 Remove the protective label from the optical lid Slide the optics module onto the U bracket 4 Secure the optics module to the U bracket using the two long thin hex screws and the hex driver provided 5 After the optics module has been installed confirm that the optics module and lid assembly can be opened and closed readily If the lid is difficult to open lower the support bracket slightly before tightening the bracket mounting screws If the lid is difficult to close try raising the support bracket slightly before tightening the rear screws 1 2 5 Connecting Power and Communication Cables to the System Before connecting any communication or power cables to the system confirm that the power switch for the iCycler thermal cycler and the optics module are in the OFF position 1 Close the optical reaction block by sliding the lid forward and pressing down on the lid handle On the right side of the optics module are three connectors 2 Using the cables provided establish power and communication with the computer as follows e Recessed 3 pin power connector Connect the supplied power cord between the optics module and a grounded power outlet This connection provides power only to the optical module a separate power cord must be connected to the iCycler thermal cycler e Serial connector A serial connector is
16. option to graph data relative to control allows you to quickly visualize upregulation and downregulation The values graphed are exactly the same Graph Data EN EN Relatve to areal 82 00 ud Quent ao Scaling Opos e 20 00 9 Ursaied a TE Hrs 7 rs 8 rers Condison Nor makred Fold Expression 6 does i Fig 6 50 Graphing Options for Displaying Gene Expression Data y Axis Options This option allows you to display the graph with the y axis in log2 or linear scale as shown in Figure 6 51 The log2 scale is useful when you evaluate samples across a large dynamic range 400 00 4 150 01 Not makred Fold Expression Fig 6 51 Options for Displaying the y axis on the Gene Expression Chart 92 Section 6 Data Analysis Module Scaling Options These options shown in Figure 6 52 are only active in Normalized Expression mode they allow you to calculate and present your data in a manner that is best suited for your experiment f Graph Date EE 1b E Tubulin Reletiveto muri Qnestiveto ne 1 509014 i 8 00E 00 4 4 20 00 4 2400 00 4 140 00 4 I mm Aa 55 xs 0p6o0g Congdon Nor makred Fold Expression Fig 6 52 Scaling options for Normalized Gene Expression e Scale to Highest This option recalculates the normalized expression for each gene by dividing the expression level of each condition by the highest expr
17. which neither contain amplification data nor End Point data may not be analyzed in the End Point tab 6 8 1 The End Point Analysis Settings The End Point tab is comprised of several sections each one described in detail below The Display Wells and Analyze Wells buttons have the same functions as described in section 6 2 4 Method Box End Point Analysis Methods Below the file and fluorophore information is the Method box The Method box allows you to select the method of assigning positive and negative values to your unknowns based on RFU values The Method box consists of the following three choices e Negatives This is the default method Select this method to use negative controls to define or call unknown samples Samples are considered positive if they are greater in RFU value than the negative control average plus the tolerance e Positives Select this method to use positive controls to define or call unknown samples Samples are considered positive if they are greater in RFU value than the positive control average minus the tolerance 75 Section 6 Data Analysis Module e Positives amp Negatives Select this method to use positive and negative controls to define call unknown samples Samples are considered positive if they are greater in RFU value than the positive control average minus the tolerance Samples are considered negative if their RFU value is less than the negative control average plus the tolerance End Poin
18. 07 0 086 1 000E 06 6 000 1 00E 06 0 00E 00 N A 4 22 42 22 46 0 056 1 000E 05 5 000 1 00E 05 0 00E 00 N A 5 25 89 25 85 0 082 1 000E 04 4 000 1 00E 04 0 00E 00 N A 6 29 07 29 03 0 130 1 000E 03 3 000 1 00E 03 0 00E 00 N A 7 33 08 32 66 0 504 1 000E 02 2 000 1 00E 02 0 00E 00 N A 1 11 55 11 70 0 193 1 000E 08 8 000 1 00E 08 0 00E 00 N A 2 15 57 15 59 0 071 1 000E 07 7 000 1 00E 07 0 00E 00 N A 3 19 03 19 07 0 086 1 000E 06 6 000 1 00E 06 0 00E 00 N A 4 22 43 22 46 0 056 1 000E 05 5 000 1 00E 05 0 00E 00 N A 5 25 75 25 85 0 082 1 000E 04 4 000 1 00E 04 0 00E 00 N A 6 28 89 29 03 0 130 1 000E 03 3 000 1 00E 03 0 00E 00 N A G09 N Std 7 32 10 32 66 0 504 1 000E 02 2 000 1 00E 02 0 00E 00 N A gt bl Amplification Data RFU Standard Curve Ct Results l4 Fig 6 11 Standard Curve C Results Spreadsheet 56 Section 6 Data Analysis Module Columns can be sorted by clicking on the column headings and reordered by clicking and dragging to move columns Data can be exported by right clicking on the spreadsheet and selecting Export to Excel Data can be printed by right clicking on the spreadsheet and selecting Print None of the data in this spreadsheet may be edited 6 4 3 Amplification Data RFU Spreadsheet You can view the individual RFU readings for each amplification trace at every cycle in the Amplification Data RFU spreadsheet Access the Standard Curve C Results spreadsheet by clicking Results on the PCR Quant screen and selecting the A
19. Carve Fit Fig 6 10 The Plate Spreadsheet The spreadsheet displays only one fluorophore at a time You can use the Plate spreadsheet fluorophore selector buttons found under the Plate spreadsheet to determine which fluorophore is displayed in the spreadsheet Each well has a colored bar at the top of the cell which is the color used in the amplification chart to display the amplification trace for this well and fluorophore The sample type is always displayed in this spreadsheet Above the spreadsheet is a set of checkboxes you can use to display the Identifier Concentration Threshold Cycle and End Point Calls in the Plate spreadsheet 6 4 2 Standard Curve C Results Spreadsheet The Standard Curve C Results spreadsheet displays the well fluorophore sample type identifier replicate number threshold cycle starting quantity and statistics for replicate groups Access the Standard Curve C Results spreadsheet by clicking Results on the PCR Quant screen By default the Standard Curve C Results tab will open displaying the spreadsheet results Figure 6 11 35 Ma4 EN Results Analysis Mode Pcr Base Line Subtracted Curve Fit x T Replicate Threshold Cycle Starting Quantity Eng Set ype Identifier ct CtMean Ct Std Dev 50 Starting SQ Mean SQ Std Dev Point Quantity 1 11 63 11 70 0 193 1 000E 08 8 000 1 00E 08 0 00E 00 N A 2 15 53 15 59 0 071 1 000E 07 7 000 1 00E 07 0 00E 00 N A 3 19 02 19
20. Click or drag across the plate to define wells with the selected fluorophore and sample type Continue defining the remaining wells that will contain the first fluorophore by changing to other sample type icons as appropriate To calculate standard concentrations automatically click Dilution Series and enter the upper or lower concentrations and units of the standards set the dilution factor and click Apply Dilution Series Repeat steps 7 11 for any additional dye layers fluorophores as required Remember that if the Whole Plate Loading box is checked changes made in standard concentrations will be applied to all dye layers for that well and extended to all replicates in the group Click Apply Plate Changes to make the changes To see the effect on analysis go to one of the other Data Analysis windows NOTE To delete a previously defined well click the Delete All icon and then click the well To delete the selected fluorophore from a previously defined well click the Delete Fluorophore icon and then click the well NOTE Use the Next checkbox to enter a particular number to assign to the next standard or sample you define NOTE The original plate setup is retained with the data file and may be restored at any time by clicking Restore Original Plate 217 7 Section 2 Introduction to the iQ5 Optical System Software Section 3 Introduction to the iQ5 Optical System Software Version 2 1 The iQ5 software is divided into
21. Data Analysis Module Gene expression analysis is the most common real time PCR application Therefore this manual uses terminology that is specific to this kind of study You can safely substitute gene with the word sequence or target when you read about how to use the gene expression module Without stringently quantified controls you cannot use this part of the iQ5 software to evaluate target concentrations of particular sequences relative to each other The iQ5 software can only evaluate relative differences of a sequence between a group of samples The gene expression analysis screen has flexible tools for the determination of the fold induction of one gene relative to another gene or relative to itself under different circumstances that is temporally geographically or developmentally different points Additionally within the gene expression analysis screen it is possible to create a Gene Study from multiple plates of data collected at different times The gene expression analysis screen Figure 6 38 consists of four main areas e The graphical data view section Displays graphed data and graph settings e The Settings and Data Table options Located to the right of the graph section Used respectively to specify analysis settings and access the data spreadsheet e The gene expression plate interface section Located at the bottom of the screen A spreadsheet view of the plate setup used to modify and edit gene and c
22. Figure 6 39 wells B4 C4 D4 are selected and are being changed from condition name 1 Hr to 1 5 Hrs The new condition name 1 5 Hrs was typed in the Condition Name box In addition to the gene expression plate interface gene and condition names can also be edited post run using Edit Plate Opening this window when analyzing collected data by clicking on the Edit Plate tab at the top of the screen will open a modified version of this window In this modified window you can edit sample type replicates standard concentrations and primer probe gene names For further details on post run edit plate functionality refer to section GeneNamel c Condition Name 1 5 Hrs HEB Copy condition to all data ses MEM Enable for Lm E 2 3 4 1 Normalized expression ddct Recalculate Relative quantity dct a n 19 93 179 15 46 19 89 17 85 15 20 I pesce D 19 90 17 90 15 49 PM Unknown Unknown Unknown 11 63 15 53 19 02 2242 25 88 29 06 33 08 Standard Standard Standard Standard Standard Standard Standard 11 55 15 56 19 02 22 43 25 75 28 89 32 10 Standard Standard Standard Standard Standard Standard Standard 11 92 15 66 19 17 22 52 25 90 29 14 32 80 Standard Standard Standard Standard Standard Standard Standard Fig 6 39 Editing Gene and Condition Names in Gene Expression Plate
23. Plates Film IQ2bEmulator IQ2bmulator xml In all cases the original calibration files with the volume dependency will be backed up to the backup folder after upgrading Section 2 Quick Guides Section 2 Quick Guides This section contains quick guides for the following topics Q o o Protocol Quick Guide page 7 Plate Setup Quick Guide page 8 Running a Real Time Experiment Quick Guide page 9 Data Analysis Quick Guides o PCR Quant Tab Quick Guide page 10 o End Point Quick Guide page 11 o Allelic Discrimination Quick Guide page 12 o Gene Expression Quick Guide page 13 o Post Run Plate Editing Quick Guide page 16 The fundamental steps of running a real time PCR experiment on the MyiQ2 MyiQ or iQ5 Real Time Detection System are the following GI ume our Da E Select Edit or Create a Protocol Select Edit or Create a Plate Setup Click Run Select the appropriate Well Factor option and begin the run Analyze the data The quick guides below outline key procedures from each step These quick guides are also located in the online help of the iQ5 Optical System software For more complete information regarding the procedures and features of the iQ5 Optical System software refer to the relevant sections of this user manual 2 1 Protocol Quick Guide Within the Workshop module click the Protocol button Selecting a Protocol 1 2 Navigate to your desired protocol file using the file tree brows
24. Setup Follow all screen prompts to finalize the installation Certain configurations of Windows operating systems initialize new folders by assigning Read and Execute permission for the members of the Users group If you have this type of operating system and this is a first time installation the administrator must change the protection for the Program Files Bio Rad folder or the Program Files Bio Rad iQ5 folder so that you can save protocol plate setup and data files If you still cannot write to the folders beneath the Program Files Bio Rad iQ5 folder after changing the protection on either of these folders check the properties of each folder Specifically in the Properties window Securities tab ensure that the checkbox that allows inheritable permissions to propagate to that folder has been selected Confirm that the iQ5 system software is working properly by double clicking on the shortcut icon located on the Windows desktop or by selecting the iQ5 program icon from the Bio Rad folder in the Windows Start menu 1 3 1 Installing the Camera Drivers Before using the real time PCR detection system for the first time the camera drivers must be installed 1 2 Power on the iCycler thermal cycler and the optics module Windows will display a Found New Hardware Wizard dialog box To install the camera drivers select the option to Install from a list or specific location Click Next to continue In the new window that appea
25. The position of the temperature bar threshold bar and the height and well of the currently selected peak are also displayed here You cannot edit any of these fields When the Edit melt peak begin end temp option is active the most recently modified temperature begin or end is displayed in the Temperature Bar field 6 6 5 Melt Curve Peak Spreadsheet The spreadsheet below the plots displays information about the melt peaks the RFU data or the d RFU dT data for each well Figure 6 27 None of the fields may be edited though you may eliminate peaks from the spreadsheet by either highlighting the peak in the spreadsheet and clicking Delete Selected Peak or by dragging the threshold bar up to a position above the peak Area Threshold Threshold Melt Peak Begin E 5 GERI PE em End Temp Area Fraction CrossingBegin Crossing End Threshold Threshold Crossing Area Crossing Area Edited Begin Edited End Temp emi Temp Temp Fraction 72 00 410 10 59 00 79 00 4636 64 7627 59 00 79 00 4636 64 78 85 59 00 79 00 90 00 224 31 86 00 95 00 1442 90 23 73 86 00 93 00 1243 71 21 15 86 00 95 00 72 00 413 50 60 00 79 00 4522 79 72 28 60 00 79 00 4522 79 7447 60 00 79 00 90 00 232 34 83 00 95 00 1734 30 27 72 83 00 93 00 1525 87 25 23 83 00 95 00 72 00 402 60 66 00 79 00 3500 89 70 24 66 00 79 00 3500 89 72 63 66 00 79 00 90 00 247 08 86 00 95 00 1483 48 29 76 86 00 93 00 1319 17 27 37 86 00 95 00 E06 E06 0 72 00 420
26. absolute quantities can be determined The efficiency of the PCR reaction can also be determined using standard curves with either known quantities used to produce the standard curve or by using a serial dilution of the template under investigation Melt Curve Peak Melt Curve Peak is a dynamic tool used to measure the melting temperature Tm of double stranded DNA molecules DNA duplexes can be visualized by either incorporation of DNA binding dyes for example SYBR Green I or by hybridization with fluorescently labeled probes Three major applications for Melt Curve Peak are peak identification number of amplified products characterization of molecular beacons and allelic discrimination End Point The End Point tab provides a convenient method of analyzing final RFU relative fluorescence unit values This can be useful when PCR Analysis is to determine if a given sample is positive or negative for a particular nucleic acid sequence Allelic Disc The Allelic Disc tab is useful for assigning genotypes to unknown samples by making comparisons to known genotypes It can be used to distinguish among homozygous wild types homozygous mutants and heterozygous individuals based either on threshold cycle or on RFU Allelic Discrimination analysis can only be performed on datasets that contain multiplexed PCR data 47 Section 6 Data Analysis Module e Gene Expr The Gene Expression screen has flexible tools for the determination of the
27. and vessel type Enter or edit a name for the experiment Click Select Add Fluorophores and select or edit the fluorophores to be used on the plate For most experiments leave the Whole Plate Loading box checked e With Whole Plate Loading changes made to any fluorophore within a well are extended to the other fluorophores within the well and within the replicate group e When Whole Plate Loading is deselected no assumption is made about the sample type in the second and subsequent fluorophores The sample type can vary from fluorophore to fluorophore and if there are standards you can define individual dilution series for individual fluorophores Click a sample type icon Select the type of replicate loading desired 23 Section 4 Workshop Module 9 Click a fluorophore 10 Click or drag across the plate to define wells with the selected fluorophore and sample type 11 Continue defining the remaining wells that will contain the first fluorophore by changing to any other sample type icons required To calculate standard concentrations automatically click Dilution Series and enter the upper or lower concentrations and units of the standards set the dilution factor and click Apply Dilution Series 12 Repeat steps 7 11 for any additional dye layers fluorophores as required Remember that if the Whole Plate Loading box is checked changes made in standard concentrations will be applied to all dye layers for that well an
28. background calibration Next perform persistent well factor calibration 7 4 3 Generating Persistent Well Factors Before you perform a persistent well factor calibration run complete the following steps 1 Prepare an external well factor plate see section 7 3 2 Ensure that the mask has been aligned and that background calibration has been performed 3 Place the external well factor plate in the iCycler cycler 4 Select the Well Factors tab in the Calibration module The Well Factor window is shown in Figure 7 4 115 Section 7 Calibrating the Instrument To generate persistent well factors 1 2 3 4 NOTE Select a well sealing type film or caps from the drop down list Select a vessel type plates or tubes from the drop down list Click Collect Persistent Well Factors When the iQ5 system completes the persistent well factor calibration run a dialog box appears with the message Persistent Well Factor Calibration Run Complete Click OK to exit If you wish to calibrate for more than one vessel and sealing type combination repeat the process above to collect additional external well factor files During a run the software will automatically use the correct file for the vessel and sealing parameters specified in plate setup This completes the generation of persistent well factors This completes calibration of cameras on the MyiQ system To calibrate the cameras on the MyiQ2 and iQ5 systems pe
29. baselines for each individual trace and the threshold that is set for the fluorophore to set conditions to determine the threshold cycle Baseline cycles and threshold are only calculated by the software when in PCR Base Line Subtracted or PCR Baseline Subtracted Curve Fit mode You can override the automatic conditions set for baseline and threshold in the Base Line Threshold window 1 Ensure that only one fluorophore from the Amp Chart Fluorophore Selector buttons is selected 2 Right click on the amplification chart 3 Click Base Line Threshold After you click Base Line Threshold in the amplification plot menu the Base Line Threshold Parameter window appears and displays the fluorophore of the traces to be adjusted 4 Select User Defined In Figure 6 15 the Base Line Threshold Parameter window shows the FAM traces are to be adjusted Wa Base Line Threshold Parameter FAM Base Line Cycles Auto Calculated User Defined Well FAM FAM FAM FAM FAM FAM Select All Edit Range Indicates changed value M Crossing Threshold Q Auto Calculated Threshold Position User Defined pois OK Cancel Fig 6 15 The Base Line Threshold Parameter Window 59 Section 6 Data Analysis Module Start and Ending Cycle Mode To change the baseline range for all wells click Select All Then click Edit Range to enter the start and end cycle for baseline calculation Figure 6 16 To change
30. by clicking on the button to make well identification and selection easier Highlight the wells in the gene expression plate interface you wish to edit e Change the gene assignments in these wells by typing your desired name into the gene pull down men then click enter to apply the name to the selected wells e Change the condition assignments in these wells by typing your desired name into the condition pull down menu then click enter to apply the name to the selected wells NOTE The Gene and Condition Names have a character entry limit of 15 characters 14 Section 2 Quick Guides A n e Minimize the Gene Expression Plate interface by clicking on the button to return to the standard view of the Gene Expr tab window Click the Settings tab then select Gene List to set your desired reference gene s Click Normalized Expression ddCt Click Recalculate to see your results E a OV Normalized expression results are graphed The Data Table spreadsheet is accessed by clicking Data Table The Data Table spreadsheet lists the Condition and Gene name calculated expression values and C values Right click to print or export this data to Excel NOTE Use the Settings tab then select Gene List to enter a specific user defined gene reaction efficiency Use the Settings tab then select Condition List to select a particular sample as a control sample If you need to compare these data to results obtained in other opd fi
31. checkbox es in the Ctrl column within the control condition s to which you want to assign a control condition 6 9 4 3 Data Set List The Data Set List displays pertinent information about the data being analyzed in a given Gene Study or opd file The information contained within the cells of the Data Set List is for display only and cannot be modified Each row of the Data Set List is linked to a single data tab of the gene expression plate interface located at the bottom of the Gene Expr window Selecting a row from the spreadsheet automatically toggles to its representative dye layer data tab in the plate interface Selecting a dye layer data tab results in toggling to its representative row in the Data Set List spreadsheet Information displayed in the Data Set List includes e Name Displays the fluorophore name for a given set of collected data A number is placed before the abbreviated fluorophore name The assigned number helps to distinguish between data from the same fluorophore when multiple files are used as in a Gene Study e Full Name This cell contains the original name of the opd file as well as the original location of the opd when it was created e Created Date This cell displays the date and time the opd file was created The Name and Full Name column contents can be alphabetically sorted by clicking on the title header of each cell This feature is helpful when long list of fluorophores are created as with multipl
32. conditions are met an algorithm is used to calculate the pair wise differences between the C values dC for all samples that qualify as inter run calibrators that is having the same Condition Name per gene and per assay Inter run Calibration Algorithm All data within the Gene Study is normalized to the inter run calibrator that yields the smallest average dC value The inter run calibrator with the smallest average dC value becomes the 103 Section 6 Data Analysis Module dominant inter run calibrator The average dC value of the dominant inter run calibrator will be used to adjust all Cy values within the Gene Study To find the dominant inter run calibrator the iQ5 Inter run Calibration algorithm first calculates the average of the dC values for all inter run calibrators of a given gene The iQ5 software uses a multi tiered algorithm to determine the dominant inter run calibrator The algorithm utilizes the following hierarchy for determining the dominant inter run calibrator 1 Setthe dominant calibrator to the gene with the highest number of common replicate groups in a given pair wise comparison If multiple genes have the same number of common replicate groups then 2 Set the dominant calibrator to the gene with the smallest range of dC values in pair wise comparisons The range is examined by comparing the absolute value of the difference between the maximum and minimum dC for the inter run calibrators of a gi
33. desired position within the list of names More than one Gene or Condition Name can be selected highlighted by clicking on the desired names Note that when the Manual order radio button is active a total of four arrow buttons are displayed The outermost arrow boxes move the selected items to the top or bottom of the list while the innermost arrow buttons allow for stepwise movement of the Gene or Condition Name e When finished sorting click OK to view the changes to the chart Sort Options Groups Actin Gapdh Gene Names Lib T Condition Names an Options Alphabetic order Manual order OK Cancel Fig 6 56 The Sort Options Dialog Box Corrected Std Devs The Corrected Std Devs selection enables the display of error correction propagated from the standard curve data This option should only be used if a standard curve was created as part of the gene expression analysis The resulting error correction is displayed as a property of the individual error bars on the chart The exact error value can be displayed by mousing over the error bars to reveal the tool tip or by displaying the Data Table and then selecting Show Details When Corrected Std Devs is active a check mark is displayed next to its name in the gene expression chart context menu Gene Expression Chart Tool Tips Tool Tips are visible on the gene expression chart when the mouse cursor is positioned on one of the chart data bars or error bars When th
34. display at the bottom of the screen indicating that while the data can be viewed and analyzed the plate setup contains fluorophores that cannot be used to collect new data using this plate setup Similarly opening a data file collected using a MyiQ2 system while a MyiQ system is connected to the software will result in the same message Figure 4 18 A Data file is invalid Plate contains fiuorophore s that cannot be used with the current instrument Fig 4 18 Invalid Data File Error 4 5 3 Opening a Pure Dye Calibration file Opening a pure dye calibration file will not only show the pure dye fluorescent data in the PCR Quant screen but will also write this calibration file to the appropriate calibration folder This will overwrite a calibration file of the same reaction vessel seal optical camera and thermal cycler and all subsequent experimental data will be collected and analyzed with the new calibration file 4 5 4 Applying Alternate Pure Dye Calibrations Alternate RME Optical data is saved to the data file together with the appropriate calibration files For the MyiQ2 and iQ5 Real Time systems it is possible to overwrite the pure dye calibration files stored within the opd data file This feature protects you from losing valuable experimental data if for example the calibrations were conducted incorrectly or a run was completed with expired well factors To apply an alternate pure dye calibration RME file to a data file 1
35. displayed both graphically and in spreadsheet format 4 1 3 Selected Plate Setup Area Located in the bottom right of the Setup window the Selected Plate Setup window Figure 4 2 displays the details of the plate setup selected in the file browser area The selected plate setup name appears at the top of this window If you have selected a data file then the plate setup within the data file and the data file name are both selected in the iQ5 software When you select the Data File tab and a data file the iQ5 software displays the Original and Current plate setup The Original plate setup is the plate setup used to create the data file The Current plate setup is the plate setup last saved with the data file If you are looking at a data file that contains a plate setup that was edited and saved after the experimental data were collected you can revert to the original plate setup by clicking Original in the Plate box To go back to the present definition click Current None of the information displayed on the plate in the Workshop home window can be edited from this screen 20 Section 4 Workshop Module Selected Plate Setup TemplateFAM_HEXpts Edit crests New origina current Sample Volume 25ul Seal Type Film Vessel Type Plates omen ES m m m OS cr Wr rs Wr ur e AS E20 Nr re e n m S Fig 4 2 The Selected Plate Setup Area 4 1 4 Selected Data File Area Located in the top right o
36. dividing the standard deviation of the normalized expression by the normalized expression value for the highest or lowest expresser depending on which scaling option you choose 2 2 2 SD Rel Quant sample Ref 1 SD Rel Quant sample Ref 2 SD Rel Quant sample Ref n SD Normalization Factor ample s xe fo eee n Rel Quant sample Ref 1 n Rel Quant sample Ref 2 n Rel Quant sample Ref n When a control is assigned you need not perform this rescaling function on the standard deviation as illustrated below SD Normalized Expression sample Gene x 2 SD Rel Quant le G Normalized Expression eo Rel Quant sample Gene x sample Gene x SD Normalization F actor sample 2 Normalization Factor sample Standard Deviation of the Scaled Normalized Expression Rescaling this value is accomplished by dividing the standard deviation of the normalized expression by the normalized expression value for the highest or lowest expresser depending on which scaling option you choose s SD Normalized Expression SD Scaled Normalized Expression sample Gene x P sample Gene x Normalized Expression way or min Gene x When a control is assigned you need not perform this rescaling function on the standard deviation 6 9 9 Relative Quantity Calculations Relative Quantity Calculations with No Controls Identified Relative Quantity dCT for any sample for gene x is ca
37. e Expression Standard Deviation The new columns are named e Corrected Rel Quant SD e Corrected Unscaled Expression SD e Corrected Expression SD EH Setting NEN DataTable EB Show Details Corrected T DataSet Condition Gene Ctrl Rel Quant Rel Qut se qun Easel poression Lime Expression Press i Beta Actin 1 00000 0 01254 0 01254 N A N A E N A N A N 2 LSYBR2 Ohr GAPDH 1 00000 0 03261 0 03261 N A N A N A N A N 3 1 SYBR3 Ohr IL1 Beta 1 00000 0 04909 0 04909 1 00000 0 05211 0 05211 1 00000 0 052 4 1 SYBR4 Ohr Tubulin x 1 00000 0 03127 0 03127 1 00000 0 03582 0 03582 1 00000 0 035 5 1 SYBRI ihr Beta Actin 0 94050 0 02513 0 02513 N A N A N A N A N 6 1 SYBR2 thr GAPDH 0 94374 0 16148 0 16148 N A N A N A N A N 7 1 SYBR3 thr IL Beta 0 25112 0 00613 0 00613 0 26655 0 02398 0 02398 0 26655 0 023 8 i SYBR4 thr Tubulin 3 80008 0 14045 0 14045 4 03354 0 37976 0 37976 4 03354 0 379 9 1 SYBRI 2hr Beta Actin 107182 0 02117 0 02117 N A N A N A N A N 10 1 SYBR2 2hr GAPDH 0 99272 0 09249 0 09249 N A N A N A N A N 11 1 SYBR3 2hr ILi Beta 0 06578 0 00759 0 00759 0 06377 0 00796 0 00796 0 06377 0 007 12 1 SYBR4 2hr Tubulin 15 79305 0 89552 0 89552 15 31059 1 13372 1 13372 15 31059 1 133 Fig 6 48 The Gene Expression analysis Data Table Show Details view A difference between corrected and non corrected values will only be seen if a standard curve was created as part of the real time PCR assay The e
38. entry limit of 15 characters 85 Section 6 Data Analysis Module To access the gene list 1 Make sure the Settings button is activated If not click Settings See the example in Figure 6 42 2 Click Gene List The list of genes appears in the spreadsheet directly below the Gene List option button as shown in Figure 6 42 JEN Seno Mm Data Table Gene List Condition List Data Set List Show Auto Efficiency Name Full Name Ref Color Graph Efficiency 96 1 Actin Actin lv mm v 96 4 2 Gapdh Gapdh v EE v v 96 2 3 ILib IL1b B EE v v 96 8 4 Tubulin i Tubulin C EE il v 95 0 Fig 6 42 Selecting the Gene List Option Assigning a Reference Gene To assign a reference gene 1 With the Settings button active select Gene List 2 Select the checkbox in the Ref column within the control condition s to which you want to assign a reference gene You can assign the role of reference gene to as many genes as you wish In Figure 6 43 Actin and Gapdh have been selected as reference genes Gene List Condition List Data Set List Name Full Name Ref Color 1 Tubulin Tubulin 2 Actin Actin v 3 IL1b IL1b E Gapdh Gapdh EN Fig 6 43 Assigning a Reference Gene 86 Section 6 Data Analysis Module Reaction Efficiency and Gene Expression Analysis Efficiency describes how much of your sequence of interest is being produced with each cycle An efficiency value of 100 m
39. file to the Gene Study once again 100 Section 6 Data Analysis Module 6 10 2 Creating a Gene Study File A Gene Study file must contain data from at least two different opd files The process of creating a Gene Study file is a 2 step process STEP 1 Prepare opd files for gene expression analysis 1 In the PCR Quant tab establish the desired experimental conditions such as baseline and threshold and the wells to be included in the analysis NOTE Additional wells can be excluded or re included later from the gene expression plate interface 2 Click on the Gene Expr tab Make sure that all files to be included in the Gene Study have the Enable for Gene Study button selected The default setting is that this button is not enabled After enabling the file for multi file analysis save the opd file 4 Repeat steps 1 3 above for all additional files to be included in the Gene Study STEP 2 Add files to a new Gene Study 1 From the menu toolbar select File gt New gt Gene Study The Gene Expression Study Manager will be displayed 2 Select Add opds The Windows file explorer dialog box will appear 3 Locate and select the opds needed for the Gene Study Multiple files may be added to the Gene Study at one time 4 Click OK when finished Once the files are added to the Gene Expression Study Manger selecting OK opens an unsaved Gene Study file in the Gene Expression window of the iQ5 software The Gene
40. five sections called modules Icons representing each of the modules are always shown on the left side of the screen The active or selected module has an orange background whereas unselected modules have a gray background Each module is subdivided into windows that perform a specific function for that module The five modules in the iQ5 software are the HN n Workshop Run Time Central Data Analysis 2 E D j Calibration User Profile Workshop Module This module is used to select a Plate and Protocol and Run an experiment It is also where experimental files are selected and opened for Data Analysis The Workshop module consists of a Setup and Plate Summary window The Protocol Plate Run Set and Data File tabs can be used to select open edit or create files In Setup the selected Protocol and Plate Setup can be run or the selected data file can be opened for analysis in Data Analysis Run Time Central Module This module is used to initiate and monitor experimental runs It is accessed from the Workshop module once the Protocol and Plate Setup have been chosen by clicking Run or Run End Point Data Analysis Module This module contains a suite of tools enabling you to conduct thorough and varied analyses of your experimental data Within this module are screens for Quantitative Melt Curve Peak End Point Allelic Discrimination and Gene Expression analyses The Edit Plate screen permits post
41. hand side of the optics module To access the emission filter wheel remove the plug from the slot located at the top of the instrument 5 Turn the filter wheels to the desired positions using the supplied ball end hex driver As long as the power to the optics module is off the filter wheels may be turned freely in either direction 6 To remove a filter grasp it on both sides with the filter removal pliers and squeeze the tab in gently pull the filter up and out 7 To insert a filter grasp the filter with the pliers and insert it into a vacant slot For the excitation filters the tab on the filter should face toward the front of the instrument For the emission filters the tab on the filter should face the right of the instrument Be sure that every position in the filter wheel has either an excitation or emission filter or a filter blank before powering on the system 8 After the filters or filter blanks have been inserted replace the rubber plugs over the slots of the filter wheels 9 Realign the tabs on the front end of the optics module cover with the tabs on the main housing Lower the cover until the top half of the camera housing snaps into place 10 Replace the screw in the rear of the optics module to secure the casing 9 2 3 Cleaning the Filters Each optics module is shipped with the specified filters pre installed ready for use Normally you should not have to reconfigure or replace the optical filters that come w
42. iQ5 Principal iQ5 Operator and iQ5 Guest have read only access to the Administration window where they can review permission settings for their role 128 Section 8 User Profiles 8 2 1 Adding New Users Users are added using the Defining Users spreadsheet Figure 8 10 MN User Preferences Administration Defining Users Save User Changes User Name Full Name Role eMail Password Delete admin Administrator iQ5 Administrator v nee O clopez iQ5 Principal v e oO ksmith iQ5 Operator v L Russell Lab iQ5 Principal v Summer Students iQ5 Guest v v Fig 8 10 The Defining Users interface of the User Profile Module User Name REQUIRED The User Name is a set of alphanumeric characters that uniquely defines each user It can be up to 15 characters long and composed of upper and or lower case characters Full Name optional Use this field to specify the full name corresponding to the User Name Role required There are 4 roles in the iQ5 Software Administrator Operator User and Guest Each of these Roles gives users within that role permission to access specific features and functions of the software Permissions granted to all of the Roles with the exception of Administrator can be customized by the Administrator eMail optional The eMail cell is an informational area for contact information Password optional The password can be any combination of letters numbers or s
43. of data points used in determining the RFU value and you can choose to select this percentage from either the beginning or the end of the cycle or from a window in the middle of the cycle 57 Section 6 Data Analysis Module Set Data Analysis Window Locate Data Analysis Window Beginning of cycle eEnd of cycle Set Window Width pe H set Window Center andwidth e um Use Full Cycle Scan Cancel Fig 6 13 The Set Data Analysis Window To set the Data Analysis Window 1 Right click on the PCR amplification plot and click Set Data Analysis Window in the menu 2 Click either the Beginning of Cycle or End of Cycle option to select data from the beginning of the cycle or the end of the cycle respectively 3 Set the window width in the Set Window Width scroll box 4 To use all data points in the cycle select Use Full Cycle Scan 5 Click OK to return to the PCR Quant screen NOTE To center the analysis around the data collected with a window in the middle of the cycle select Set Window Center and Width Use the up or down arrows of the Set Window Width scroll box to select the percentage of data points These data points will be chosen around the value set in the Set Window Width Full Cycle text box Click OK to return to the PCR Quant screen Digital Filter After you click Digital Filter in the amplification plot menu the Set Digital Filters window appears Figure 6 14 Th
44. optical data file with a different name from the original to ensure that the original file with the original well factor calibration data is maintained for reference NOTE Do not use the well factor file containing dynamic well factors identified by the name Dynamic_ since this file contains only the well factors for the wells that were used in the experiment that generated this file If the well list does not match the operation will fail Hence it is a good practice to only use the Persistent Well Factor files when applying alternate well factor 40 Section 5 Run Time Central Module Section 5 Run Time Central Module This section contains information of the following topics o Initiating a Run page 41 o Well Factors page 42 o Real Time PCR experiments using binding dyes page 43 o Running End Point experiments page 43 o Monitoring a Run page 45 o Run Time Protocol Editing page 45 The Run Time Central module is entered automatically after clicking Run or Run End Point from the Workshop Setup window Figure 5 1 There are three tabs within the Run Time Central module Initiate Run Show Plate and Monitor Run E Setup Wl Plate Summary EN Protocol MEN Plate EN RunSet NEN Data File Selected Data File MyiQ Gene Expression SYBR Datal opd Masks fjar MyiQ 24old Dilution Data C3 Pemistem ar MyiQ 96well FAM Uniformity Data ReportTe i MyiQ 96well SYBR Uniformity Data SYBR Gene Exp
45. plot and vice versa If you place the cursor over a trace so that the pointer turns into a hand the trace will be identified in the small box placed in the top right corner of the plot The chart can be expanded to fill the window by clicking the box in the top left corner of the plot Once expanded it can be returned to its original size by clicking the box in the top left corner 65 Section 6 Data Analysis Module Melt Curve Chart 70 Temperature Celsius Fig 6 22 Melt Curve Chart The data in the melt peak chart are derived from the melt curve chart Figure 6 23 Melt Peak Chart d RFUydT 70 80 Temperature Celsius Fig 6 23 Melt Peak Chart The data in the melt peak chart show the negative rate of change in fluorescence with changing temperature that is d RFU dT Where T is temperature In analyzing the melt peak data the software assigns Begin and End temperatures to each peak and then calculates an area beneath that curve The floor of the peak area is specified by the position of the threshold bar In order to be identified as a valid peak a peak must have a minimum height relative to the distance between the threshold bar and the height of the highest peak Therefore if the threshold bar is dragged downward increasing the distance between the threshold bar and the highest peak previously unidentified peaks may show up in the
46. protocol and plate setup useful when you repeat the same experiment on a regular basis If the Run Set tab is selected the iQ5 software displays run set files with the extension run The selected run set which is a linked protocol and plate setup is displayed in the Selected Protocol and Selected Plate Setup windows e Data File Primarily used to select and open a data file You can also use this tab to run a real time experiment using the same protocol and plate setup that were used to create the data file The selected plate setup can be either the Original or Current last saved plate setup If the Data File tab is selected the iQ5 software displays data files created from previous experimental runs which have the file extension opd The iQ5 software displays the selected data file name and any associated notes in the Selected Data File window The Notes box is editable only after you open the data file The iQ5 software displays the protocol and plate setup used in creating this data file in the Selected Protocol and Selected Plate Setup windows respectively 4 1 2 Selected Protocol Area Located in the bottom left of the Setup window the Selected Protocol area displays the details of the protocol selected in the file browser area The selected protocol file name appears at the top of this window If a data file is selected the iQ5 software displays the protocol used to create the data file and the data file name The selected protocol is
47. setpoint temperature then pause at that step Clicking Pause Stop Run activates two new buttons Click Resume Run to resume the thermal cycling protocol Click End Run to terminate the experiment 46 Section 6 Data Analysis Module Section 6 Data Analysis Module This section contains information on the following topics o o o PCR Quant tab page 48 Amplification chart page 49 Standard Curve chart page 53 Results section page 55 Melt Curve and Peak charts page 65 Allelic Discrimination module page 69 End Point analysis tab page 75 Gene Expression analysis tab page 78 Gene Study Multi file Gene Expression Analysis page 99 Post Run Plate Editing page 105 Reports page 107 The Data Analysis module is where data is presented and analyzed When the iQ5 software opens the Data Analysis module is gray out and inactive To analyze a data file open the file in the Data File tab of the Workshop module by selecting the file and then clicking Analyze The Data Analysis module consists of six tabs PCR Quant The PCR Quant tab is used to set the analysis conditions for the data file The analysis conditions include setting the PCR baseline and threshold and the wells to be excluded or included in the experiment The analysis conditions should be set before using the Gene Expression End Point or Allelic Discrimination tabs For experiments with standards of known quantities the PCR Quant tab is also where
48. specific user defined gene reaction efficiency NOTE Use the Settings tab then click Condition List to select a particular sample as a control sample 6 9 3 Specifying Gene and Condition Labels for Gene Expression Analysis Use the gene expression plate interface to edit gene names for example actin GAPDH and experimental conditions names for example treatment types time course points etc Use the Settings option to select which of these genes and or conditions to use for normalization and set other analysis and display parameters Using the Gene Expression Plate Interface to Edit Gene and Condition Names Open a opd Real Time PCR Data file 82 Section 6 Data Analysis Module In the PCR Quant screen assess threshold and baseline for the data file and then make changes if necessary Click the Gene Expr tab Highlight and select the wells to be edited in the gene expression plate interface NOTE Expanding the gene expression plate interface view by clicking on the button makes the entire plate area visible for easier well identification and selection Change the gene and condition assignments in these wells by typing in the gene and condition pull down menus then click Enter to apply the name to the selected wells Entered names will then be added to the pull down menu and become available for selection from this menu NOTE The Gene and Condition Names have a character entry limit of 15 characters In
49. spreadsheet as their height becomes significant in relation to the highest peak Similarly dragging the threshold bar up can cause previously identified minor peaks to drop off the spreadsheet 66 Section 6 Data Analysis Module The vertical temperature bar may be dragged to any position on the plot The temperature bar on the melt curve plot moves along to the same position as the temperature bar on the melt peak plot is moved If you place the cursor over a trace so that the pointer turns into a hand the trace will be identified in the small box placed in the top right corner of the plot The chart can be expanded to fill the window by clicking the box in the top left corner of the plot Once expanded it can be returned to its original size by clicking the box in the top left corner 6 6 1 Melt Curve and Melt Peak Chart Menu There are a number of features of the plot that may be modified as well as control of data filtering All changes specified by the context menu accessed on the melt peak plot are also carried out on the melt curve plot Those features are accessed by a right click on the chart which brings up the menu shown in Figure 6 24 Digital Filter Adjust Graph t Define Trace Style Restore Graph Show All Traces Copy Graph Print Data Print Graph Fig 6 24 Melt Curve Peak Chart Context Menu 6 6 2 Delete Selected Peaks To remove a peak from the analysis highlight the peak in the spreadsheet a
50. step and will hold that setpoint temperature until the Continue Running Protocol button in the Thermal Cycler tab of the Run Time Central module is selected To program an infinite hold 1 Click Infinite Hold in the Show Options box A new column titled Hold appears in the spreadsheet 2 Select the Hold checkbox for the step that you want to maintain at a constant temperature and enter the desired temperature in the Setpoint cell of the spreadsheet Ramping The ramp rate is the speed with which the iCycler thermal cycler changes temperatures between the steps of a cycle or between cycles The default condition is for the iCycler thermal cycler to adjust temperatures at the maximum ramp rate The iCycler thermal cycler allows you to change temperatures at a fixed rate less than the maximum Ramp rates are adjustable to 0 1 C second and must fall within the range of 0 1 to 3 3 C per second for heating and 0 1 to 2 0 C per second for cooling Invalid ramp rate entries are adjusted to the nearest valid entry To adjust the ramp rate Click Ramping in the Show Options box A new column titled Ramp Rate will appear in the spreadsheet Double click in the Ramp Rate column on the line of the spreadsheet containing the temperature toward which you wish to control the ramp rate Use the pull down menu to select MIN or MAX or make a direct entry into the field If an invalid ramp rates is input it is adjusted to the nearest valid ramp rate
51. that are saturated will be shown in pink 4 Click Show Mask The Mask is displayed as an array of green boxes Figure 7 2 Image Fig 7 2 Camera Image with Overlying Mask Array 5 If any pink saturated pixels are present in the mask reduce the exposure time and retake an exposure Keep retaking the exposure until no pink pixels are present 6 Click Optimize Mask Each individual box should be centered over the well so that all the fluorescent signal from the well falls within the box 7 Click Save Mask to save mask data xml file in the Mask folder of the iQ5 Folder The Mask can be displayed or hidden using the Show Mask button The Mask array can be moved as a group by clicking on the Up Down Left or Right buttons An individual box within the array can also be moved by first clicking on that box to select it then using the Up Down Left or Right buttons to move it The Mask can be restored to the last saved Mask by clicking Reload Mask The default factory Mask can be restored by clicking Restore Factory Mask This completes Mask Alignment calibration Next perform background calibration 7 4 2 Performing Background Calibration Background calibration is performed to account for fluorescence in the experimental system that is due to the reaction vessel and sealing mechanism What You Will Need You need the following to calibrate the camera e 96 well PCR plate or preferred reaction vessel referred to
52. the parameters listed in the drop down menu The Sort Data By drop down menu works in conjunction with Ascending Order and Descending Order radio buttons Each of the parameters in the report can be sorted in ascending or descending order by the appropriate radio button The results of the sort are immediately displayed in the report display area e Page Setup Clicking the Page Setup button opens the Windows Page Setup dialog where different page display parameters can be adjusted e Print Preview Clicking the Print Preview button opens the Windows Print Preview screen where the printed layout of the pending report can be previewed e Print Clicking the Print button opens the Print screen where different print parameters can be adjusted and the report printed e Save to File Clicking the Save to File button opens the Save report screen where a specific location can be chosen to save the report in rich text format 6 12 2 Parts of the Data Report Each report template contains a preset report header that outlines specific information about the dataset being analyzed such as time and date of file creation and file path information At the bottom of the General Data section there is information that can be used to determine whether the report contains saved or unsaved changes to data analysis settings including e Report differs from last save The Report differs from last save report entry can be used to determine whether the analysis
53. the allelic discrimination data 9 Threshold Cycle displays the distribution of samples on the scatter plot based on the threshold cycle Cr Samples that do not cross threshold will be assigned the C value of the last cycle run in the experiment RFU displays the distribution of samples on the scatter plot based on the RFU generated by each sample at the last PCR cycle number Click the drop down list next to the Select Cycle box to generate the scatter plot based on an RFU from a different cycle of the PCR experiment Click Automatic Call or Manual Call Automatic Call is the default parameter In Automatic Call threshold bars are positioned automatically in one of two ways Based on distribution of the control wells when at least three wells have been assigned to Control 1 and three to Control 2 At 90 percent of the Cr range or 10 percent of the RFU range on each axis if no controls are named Manual Call is the alternative analysis mode Adjustments may be made either in the scatter plot or in the data spreadsheet Scatter plot Make a selection in the Allele Call box Then click and drag the cursor over the corresponding sample s in the scatter plot The iQ5 software reassigns the samples to the allele call selected by the radio button and updates the data spreadsheet accordingly Data spreadsheet Click in the Call cell of the spreadsheet A menu appears that lets you select an allele call for that sample Once you select
54. the baseline for individual wells click on a well and then click Edit Range to enter the start and end cycle for baseline calculation Edit Both start and ending cycle F Start Cycle B E Ending Cycle 5 ES DK Cancel Fig 6 16 The Both Start and Ending Cycle Mode Start Cycle Only Mode By default both the start and ending cycle for baseline calculation are altered If you select the Start cycle only mode you can only edit the Start Cycle box This mode is useful if you want to retain the automatically determined end cycle but wish to change the value of the start cycle Enter the desired value for the start cycle for the selected traces and then click OK Figure 6 17 Select individual wells or all wells as described above Edit Start cycle only e Start Cycle B d Ending Cycle ES DK Cancel Fig 6 17 The Start Cycle Only Mode Ending Cycle Only Mode In the Ending cycle only mode you can only edit the Ending Cycle box This mode is useful if you want to retain the automatically determined start cycle but wish to change the value of the end cycle Enter the desired value for the end cycle for the selected traces and then click OK Figure 6 18 Select individual wells or all wells as described above 60 Section 6 Data Analysis Module Edit Ending cycle only x Start Cycle u Ending Cycle 5 uc OK Cancel Fig 6 18 The Ending Cycle Only Mode Setting the Threshold Manually By default the iQ5
55. then click OK NOTE Two or more different dyes may be used with the same filter pair however two dyes using the same filter pair may not be used in the same well Select new Sample Volume Seal Type and Vessel Type from the drop down menus as appropriate for this experiment then click Save amp Exit Plate Editing Choose a meaningful file name and save the Pure Dye Plate Setup IMPORTANT Pure dye calibrations are made for each instrument dye type sample volume seal type and vessel type and are instrument and experiment specific Do not attempt to use pure dye calibration results on a different instrument seal or vessel type or sample volume 7 5 Viewing Calibration Files Background and well factors calibration files are saved to their respective folders within the iQ5 program folder Pure dye calibration files are saved in the RMEData folder in the iQ5 program folder Previous calibrations are saved in the subdirectory called Backup It is advisable to periodically backup your calibration files by copying the calibration folders and their contents 119 Section 7 Calibrating the Instrument To determine what fluorophore dyes an instrument has been calibrated for within the iQ5 software select Calibration Data from the View menu Figure 7 9 to open the Calibration Data file view Fluorophore dyes for which the instrument has been calibrated will be listed by name in the summary table View Reports Tools Applica
56. 38 56 00 80 00 5206 20 74 91 56 00 80 00 5206 20 77 34 56 00 80 00 E06 1 90 00 252 15 84 00 95 00 1743 86 25 09 84 00 93 00 1525 55 22 66 84 00 95 00 M 4 MJ Melt Peaks RFU d RFU dT 4 Fig 6 27 The Melt Peaks Spreadsheet 68 Section 6 Data Analysis Module The spreadsheet displays the following information for each peak e Peak ID A unique identification number in the format RCC N where R is a row letter CC is a column number and N is a number beginning with 0 1 2 etc In Figure 6 27 the first peak for well E3 is identified as E03 0 and the second peak as E03 1 e Melt Temp The temperature at the highest point of the melt peak e Peak Height The highest point of the melt peak e Begin and End Temp Starting and ending point for melt peak Area calculations are based on these starting and ending points These values may be edited but not in the spreadsheet e Area The area beneath the melt peak curve bounded by the default peak begin and end temperatures and the default position of the threshold bar Because this area calculation is defined by the default values of begin and end temperature and threshold bar it does not change as the threshold bar is moved or if the begin or end temperatures are edited e Area Fraction 9 o When there is more than one peak associated with a well the contribution of each well to the total area beneath all melt peaks for that well is calculated The iQ5 software makes this calcula
57. CR Kit for Probes 50 x 50 pl reactions iScript OneStep RTPCR Kit for Probes 200 x 50 ul reactions 142 Life Science Group 10005604 Rev C US EG Bio Rad Laboratories Inc Web site www bio rad com USA 800 4BIORAD Australia 61 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 3237 9400 Canada 905 364 3435 China 86 21 6426 0808 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 840 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000 08 0954 0109 Sig 0308
58. Cr location thus affecting all subsequent data analyses that depend on C values This would include standard curve calculations and quantification of unknowns gene expression and allelic discrimination using threshold crossing values In addition removing wells from analysis will change the statistics for replicates To select the wells to include in data analysis 1 Inthe PCR Quant tab click Analyze Wells The Select Wells to Analyze floating window appears A set of fluorophore selector buttons appears at the top of this window When you click a fluorophore button the sample type present in that well for that fluorophore is colored 2 To select or deselect an individual well click inside that well Wells included in the analysis appear with a yellow and black border Wells excluded from the analysis appear with a pale border When a well is excluded from analysis all fluorophores in that well are excluded To select all wells click Select All 4 To toggle the current selection so all wells currently selected will be unselected and vice versa click the uppermost left cell of the spreadsheet this cell has the color of the currently selected fluorophore You can also perform this action on a subset of the spreadsheet by clicking the appropriate row or column header Only the wells in that row or column are toggled 5 After you determine which wells to include in analysis click Apply if you want the Select Wells to Analyze wind
59. Data is selected the results spreadsheet displays two tabs the Amplification Data RFU tab and Standard Curve C Results tab You can perform the same function by clicking Results 63 Section 6 Data Analysis Module Restore Graph Option Click Restore Graph to redraw the graph to its original size after you zoom in on the graph Show All Traces Option Click Show All Traces to show all traces after you single out one or more using Display Wells or by clicking on a trace in the amplification plot 6 5 3 Data Export Options The amplification plot menu offers four additional options for printing and exporting graphs or data Copy Graph Click Copy Graph to copy the amplification plot into the clipboard to import into other programs Print Graph Click Print Graph to print the graph to your default Windows printer Print Amplification Data The Print Amplification Data option is only available when the iQ5 software displays the Amplification Data RFU spreadsheet You can display this spreadsheet by clicking Results in the PCR Quant tab or by clicking Display Data in the amplification chart menu When you click Print Amplification Data the Print Preview dialog box appears so you can print the amplification data Print Std Curve Data The Print Std Curve Data option is only available when the iQ5 software displays the Standard Curve C Results spreadsheet You can display this spreadsheet by clicking Results in the PCR Quant tab o
60. Expression Study Manager The columns and rows of the Gene Expression Study Manager Figure 6 59 are adjustable by clicking holding and dragging the column row separator lines The overall size of the Gene Expression Study Manager box can also be resized by clicking holding and dragging the outermost horizontal or vertical edge The Notes section located at the bottom of the Gene Expression Study Manager allows for input of text details pertaining to the study Clicking Show Details located below the opd spreadsheet displays two additional columns of information The two additional columns provide information about the Cycle and Step used for data collection and analysis in the source opd file F Gene Expression Study Manager Add or Remove OPD files L1 File Name File Directory Created Date lt i SYBR_Timecourse1 C Program Files Bio Rad iQ5 Users admin 11 11 2008 SYBR_Timecourse2 C Program Files Bio Rad iQ5 Users admin 11 11 2008 Remove SYBR Time course experiment Beta Actin GAPDH Tubulin Il 1Beta Fig 6 59 Show Details view of the Gene Expression Study Manager Window 6 10 4 Editing a Gene Study A minimum of two different opd files are required to create a new Gene Study file Additional opd files may be added to an existing Gene Study file by clicking the Edit Study button from within the Gene Expr window The Edit Study button is only displayed in the Gene Expr window when a Gene Study fi
61. Gene Expression Plate Interface e Copy Gene Name and Copy Condition Name These two commands are useful shortcuts for copying and pasting gene and condition names across many different samples within a single opd or Gene Study 84 Section 6 Data Analysis Module 1 Activate the copy command by first selecting the wells to be copied from the plate interface 2 Right click on the plate interface to reveal the Copy Gene or Copy Condition options and select the desired option 3 Once the Copy Gene or Copy Condition option has been selected the Paste Gene Condition Name text from the menu becomes active no longer grayed out 4 Return to the plate interface to select the destination wells for the copied identification information 5 Right click on the plate interface to reveal and select the Paste Gene Condition Name option e Show Sample Type The Show Sample Type option is selected by default This selection is illustrated by a check mark shown before the text in the menu Clicking on Show Sample Type toggles the display of the Sample Type in the plate interface Choosing to remove the sample type information from the plate interface simply allows visibility of a greater number of wells in the plate interface grid e Print The Print command on the plate interface menu will print the displayed plate interface e Export to Excel The Export to Excel command is useful for exporting exact values from the plate interface W
62. HB Show error details Cause Pure dye calibration data not found This error occurs when a run start is attempted using a fluorophore with a vessel and seal combination the instrument has not been calibrated for Solution The instrument should ideally be re calibrated for the fluorophore with desired vessel and seal type combination This is important for correct dye de convolution in multiplex experiments NOTE If a run must be started immediately select a vessel and seal type for that fluorophore for which the instrument has already been calibrated After the run has completed recalibrate the instrument for the desired vessel and seal combination and back apply this alternate pure dye calibration RME file to your data file Refer to section 4 5 4 for further details 139 Appendices xl o Failed to open Protocol File The Following file G temp 1 tmo is saved in an unsupported Format and cannot be opened Cause Bad protocol file There is no step defined for data collection or analysis in the thermal protocol Solution At least one data acquisition step must be present in the thermal protocol Open the protocol editor and specify a data collection at one step of the protocol A data collection step is indicated by a yellow real time data collection or green melt curve data collection camera icon at the appropriate step in the graphical display of the protocol Refer to section 4 3 for further details
63. Interface Copying Conditions to All Data Sets The Copy conditions to all data sets button should be activated if you want all fluorophores or data sets to have the same Condition Name s assigned to selected groups of wells This is a useful function if 83 Section 6 Data Analysis Module e You have a multiplex experiment In this case all sample types in the varying fluorophores must be the same Or e You are importing multiple opd data files into a Gene Study Make sure the Copy conditions to all data sets button is highlighted active when you assign condition sample names as shown in Figure 6 40 Active is the default state for this button Gene Name IL ib Condition Name Copy condition to all data sets Enable for Gene Sudy Analyze Wells 1 2 3 4 5 6 7 8 12 60 15 91 19 23 22 69 26 03 29 42 Standard Standard Standard Standard Standard Standard Fig 6 40 Copying Gene and Condition Settings to all Fluorophores Gene Expression Plate Interface Context Menu The plate interface menu is displayed by right clicking on the plate interface When this action is performed a menu is displayed with the following items Copy Gene Name Copy Condition Name Paste Gene Condition Name Show Sample Type Print and Export to Excel Figure 6 41 4 19 89 Copy Gene Name Copy Condition Name 19 90 Unknown v Show Sample Type Print Export to Excel Fig 6 41 The Context Menu of the
64. Line Subtracted Curve Fit zie Section 6 Data Analysis Module The alternative to viewing the data in All Candidates mode is to view the data in Single Point mode Adjust Graph Rescale or change the amplification plot from linear to log or vice versa by selecting Adjust Graph The Chart Axes Range Definition window appears Figure 6 19 Y Max 5205 Y Mn x Mn x Max z B a oa Log View Restore Auto Scaled Chart OK Cancel Fig 6 19 The Chart Axes Range Definition Window Enter the maximum and minimum values for the x and y axes into the Y Max Y Min X Min and X Max scroll box es or use the up and down arrows in each scroll box Change the display to a semi logarithmic view by clicking Log View Click Log View again to revert to a linear plot Revert the plot settings to the default by clicking Restore Auto Scaled Chart NOTE You can also change the display to a semi logarithmic view by selecting Log View in the PCR Quant tab Define Trace Style Option You can customize the display by selecting Define Trace Style option The Define Trace Style window appears Figure 6 20 Change the trace color and symbol which are used to display data points on a well by well basis or in groups of sample types You can preview all changes before you apply them by clicking Preview 62 Section 6 Data Analysis Module Define Trace Style Negative Controls Positive Controls B No Template Control
65. Login dialog box will appear 3 Select the desired user from the User pull down list Enter the password if any in the Password text box and click OK 4 If the user name you want is not available you will need to add it in the User Profile module in the Administration tab 8 3 2 Changing Password To change the password of the current 1 Click on the Tools menu and select Change Password The Change Password window opens Figure 8 14 2 Enter the old password in the top text box Enter the new password in the New Password and Confirm New Password text boxes 3 Click OK A Change Password cog A Old Password E New Password I Confirm New Password Fig 8 14 The Change Password Dialog Box 131 Section 8 User Profiles 8 3 3 Defining Roles Defining Roles Save Role Changes __ iQ5Principal iQ5Operatr iQ5 Guest Start Pause and Abort Runs Add Repeats to a Run Perform Skip Cycles Create Gene Study Files Perform Instrument Calibration Apply Different Calibrations to a Data File Change Endpoint Run Set Point Use Expired Calibrations for Runs s S S S S SI S S S s S S S C1 D SI S S L1 L1 DJ DJ SI DJ L3 ng Fig 8 15 The Defining Roles interface of the User Profile module Figure 8 15 lists the set of features and functions permission that can be granted by the iQ5 Administrator to users assigned to one of three non adminis
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67. Print Graph Print Graph prints the graph to your specified printer in Windows e Restore Graph Restore Graph is active only after you zoom in on the chart Clicking Restore Graph returns the standard curve chart to its un zoomed state e Show Labels Show Labels labels standards and unknowns with the well name as shown in Figure 6 9 Standard Unknown 35 30 25 Threshold Cycle Log Starting Quantity copy number E 98 5 R 2 1 000 s lope 3 357 Fig 6 9 The Standard Curve Chart with Labels 6 4 Results Section The Results workbook consists of three spreadsheets e Plate spreadsheet e Standard Curve C Results spreadsheet e Amplification Data RFU spreadsheet Click Results to toggle between the Plate Spreadsheet and the Amplification Data RFU or Standard Curve C Results spreadsheets E Results 6 4 1 Plate Spreadsheet The Plate spreadsheet Figure 6 10 displays data for each well in a grid fashion that represents the plate setup used in the experiment The spreadsheet is viewable at the bottom of the PCR Quant screen and can be expanded to full screen by clicking on the button in the top left hand corner of the spreadsheet 55 SampleType SampleType B c D E semplerype LE i E Section 6 Data Analysis Module E Loa Vie Display Wells J Analyze Wells Concentration Threshold Cycle Analysis Mode End Point Calls PeR Base leis Salitracied
68. Protocol Plate Edit create New Protoco Origina Ocurrent Ld _Create New J P oria O Corer Cyde 1 Cycle 2 40X Sample Volume 25ul Seal Type Film Vessel Type Plates s Om ex 95 0 55 0 al iiis EAEE ae oss ey le oa 59 a0 a te e A a B 2 c e D i Dwell PCR Melt Data Hopes ae Time mL Acquisition E 5 i 1 1 3 00 95 0 F 5 2 40 G 1 0 10 95 0 L 2 0 30 55 0 Real Time H 5 Fig 4 17 The Run Set Selection Window 4 4 1 Selecting a Run Set 1 Click Run Set 2 Usethe browser below the Run Set button to locate the folder containing the run set file and then click on the file name to select it 3 The selected run set will appear in the Selected Protocol and Selected Plate Setup windows 4 4 2 Creating a Run Set To create a run set 1 Select the plate setup and protocol that are to be linked 2 The plate setup and protocol can be stand alone files plate setup files have the extension pts while protocol files have the extension tmo or they can be already together in a data file with the extension opd A data file has two plate setups Original the Plate Setup the data file was created with and Current the plate setup the data file was last saved with The run set will be created with the plate setup displayed in the Selected Plate Setup window 3 Once you have chosen the protocol and plate setup files from the File toolbar menu select New Run S
69. Run Calibrator Error 6 11 Post Run Plate Editing The Post Run Edit Plate window allows you to edit the plate setup of a data file after an experiment has been performed This feature allows you to make corrections for incorrect sample type identifiers conditions probe primer names units or standard concentration assignments and to add notes about the experiment You may not add or remove fluorophores from wells in post run plate editing nor may you delete a well from the plate setup by removing both its sample type and fluorophore assignments Use Analyze Wells to remove undesired wells from the analysis After editing the plate you can reanalyze the experimental data with the new plate setup NOTE You can always restore the original plate setup definitions by clicking Restore Original Plate 105 Section 6 Data Analysis Module For post run editing of the Plate Setup saved in a data file 1 From the Workshop module a Click Data File above the directory of the home workshop Navigate the directory until the desired data file is found Double click the file name to bring the file directly into the Data Analysis module or b Click Data File above the directory of the home workshop Navigate the directory until the desired data file is found Click the file name once to open the plate setup associated with the data file in the bottom right section of the Workshop window Click Analyze to bring the data to the Data Analys
70. Sample Fig 6 60 The Include Sample Exclude Sample Selection Pull Down Menu This drop down list works similar to Analyze Wells button see Analyze Wells for details To exclude wells from the Gene Study analysis select the specific wells from the plate interface Click on the drop down arrow box to select Exclude Sample from the list The highlighted wells will be grayed out to indicate that they are no longer included in the Gene Study analysis To include wells for the Gene Study analysis select the specific wells from the plate interface Click on the drop down arrow box to select Include Sample from the list The selected wells will be displayed with the correct Gene and Condition Name colors to indicate that they are included in the Gene Study analysis 6 10 6 Inter Run Calibration Inter run Calibration is unique to the Gene Study module Inter run Calibration is automatically performed by the iQ5 software in every Gene Study The resulting Inter run Calibration calculations are then used to normalize inter run variations between genes assayed in separate real time PCR runs that is different opd files The following conditions must be met for Inter run Calibration A given sample must have the exact same Condition Name on each plate per assay and for each gene this allows the sample to be designated as an inter run calibrator e At least one inter run calibrator sample must be present in the Gene Study When the above
71. Select the data file in the file browser 2 Select Apply Alternate RMEs from the Tools menu 3 Use the Select RME File Name dialog box to navigate to then open the RME calibration file 39 Section 4 Workshop Module 4 After the RME calibration file has been opened a Save Optical Data File dialog box will appear The existing pure dye calibration will be overwritten NOTE It is recommended to save the optical data file with a different name as this will ensure that the original file with the original calibration data is always maintained for reference 4 5 5 Applying Alternate Background or Well Factors If well factors have been collected incorrectly or data has been collected with expired well factors you may apply an alternate well factor calibration to a data file When selecting an alternate well factors file to apply use Persistent Well Factor files only To apply an alternate Well Factor file to a dataset 1 Select the data file in the file browser 2 Select Apply Alternate Well Factors from the Tools menu 3 Use the Select Well Factors File Name dialog box to navigate to then open the desired Well Factors calibration file 4 After the Well Factors calibration file has been opened a Save Optical Data File dialog box will appear The existing well factor calibration for this data file will be overwritten The same approach can be used to apply alternate Background Factors NOTE It is recommended to save the
72. Study which contains all the files selected from the Gene Expression Study Manager can now be analyzed as a normal gene expression file by selecting the analysis method assigning Gene and Condition Names from the gene expression plate interface and assigning attributes from the Gene List NOTE Gene Study files are NOT automatically saved at the time of file creation It is highly recommended to save the newly created gxd file after initiating a Gene Study When the files are added to the Gene Expression Study Manager the file information is separated into four columns e File Selection This column is used in conjunction with the Remove button and features a checkbox in the column header The header checkbox will select or deselect all the displayed files chosen for the Gene Study To select or deselect an individual file dick the checkbox within the corresponding row If one or more files have been selected dicking Remove will remove those file s from the Gene Study e File Name This column displays the full name of the opd files to be included in the Gene Study e File Directory This column displays the directory location of the opd files to be included in the Gene Study 101 Section 6 Data Analysis Module e Created Date This column displays the creation dates of the opd files to be included in the Gene Study Note that these dates are NOT the dates of the most recent save event for the listed files 6 10 3 Gene
73. a de aene HARE VEDRAERRRRAEXRDE UE XRRRRARATRARSRUERERRA CERA REN EARS 120 7 0 2 Came erri 120 Section 8 User Profiles ctes seen 122 8 1 User Pref rences cine teres en aana Pe i at nt need Pe DV CE YR a Eu aaa aAa 122 8 1 1 File Paths Preferences eire rne Daci REFER cena sadeeessacadedna haaa na haa ia daadaa d Esaa 123 8 1 2 Plate Setup Preferences eeeeeeeeeeseeeeeeee nennen enne nnn ener nnns sane nan anne 124 8 1 3 Protocol Preferences 2 2 cere De ER RR EE RR RERR FEL EAA a A A EE A RG 125 8 1 4 File Selection at Application Startup Preferences eene 125 8 1 5 PCR Quant Screen Preferences esses nennen nennen nennen nnn nnn nenne nnn 126 8 1 6 Allelic Discrimination Module Preferences cccccccececececececececeeeeeeeeeseeeeesaeaeeeeenes 127 8 1 7 End Point Module Preferences cccccccccecececececeeeeeceeeeeeecesecesesesesesesesanananeneenenes 127 8 1 8 Gene Expression Module Preferences eeeeeeeeeeeeeeeeene nennen nnne nennen nnns 128 8 2 User Admihistration 22i eene ia Er gap ea Er Les e ra ELE aaa GER ea La Es La oua ada 128 8 2 1 Adding N w USES 129 8 2 2 Deleting Existing SOLS ri seve taceschentvevanevandachestvedasevagebenduthevesdsiatedhcstavtecetivecchiadewentens 130 8 3 Logging on to the iQ5 Software ce i eee nnn nnne 130 8 3 1 Switching USer s ice ert ntt a aaa aaa aa DE das scc
74. a file is invalid Plate contains fiuorophore s that cannot be used with the current instrument Cause Data files collected using an iQ5 system can be opened when the computer running iQ5 software version 2 1 is connected to a MyiQ2 or MyiQ system The warning message above indicates the plate setup contains fluorophores not compatible with the connected instrument This message means that while the data can be viewed and analyzed the plate setup contains fluorophores that cannot be used to collect new data Similarly opening a data file collected on the MyiQ2 system when a MyiQ system is connected to the software will result in the same message if MyiQ incompatible fluorophores are present in the plate setup Solution If you wish to view or edit the data no corrective action is required If you want to repeat the run using the plate setup associated with the data file you will need to edit the plate setup to remove fluorophores that are incompatible with the connected instrument Refer to section 4 2 and 4 5 for further details 137 Appendices I x 2 Camera connected but could not connect to the thermal cycler 1 Please ensure the device s are powered on and properly connected then re startthe application Cause This error appears when the software computer is unable to communicate with the camera The iQ5 software and camera are on but the iCycler base unit is powered off Solution Close the software t
75. action using standard curves with either known quantities used to produce the standard curve or by using a serial dilution of the template under investigation EE PCR Quant EE Melt Curve Peak WE End Point Allelic Disc EH Edit Plate Amplification Chart Standard Unknown 407 Threshold Cycle PCR Base Line Subtracted Curve Fit RFU 25 204 i 154 2 4 6 8 10 Log Starting Quantity copy number e FAM E 99 7 R 2 0 998 slope 3 328 y int 46 e HEX E 99 2 R 2 0 998 slope 3 342 y int 46 Identifier MB Log View Display Wells Analyze Wells Concentration Threshold Cycle Analysis Mode End Point Calls Per Base Line Subtracted Curve Fit Se HERE ee ET BER std2 std 2 Unkn 2 Unkn 2 SampleType std 3 std 3 pe oes oe std Unkn a _Unkn 5 SampleType E SampleType d Unkn 5 F Sampletype eae oe G SampleType H sampletyee stds std s Std Unkn s Unkn 8 Unkn 8 ux Fig 6 1 The PCR Quant Tab 48 Section 6 Data Analysis Module The PCR Quant tab consists of three sections e Amplification chart e Standard Curve chart e Results section 6 1 1 Customizing the PCR Quant Display You can customize the size of the sec
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77. additional dye layers fluorophores as required Remember that if the Whole Plate Loading box is checked changes made in standard concentrations will be applied to all dye layers for that well and extended to all replicates in the group NOTE To delete a previously defined well click the Delete All icon and then click the well To delete the selected fluorophore from a previously defined well click the Delete Fluorophore icon and then click the well 13 Click Save amp Exit Plate Editing 2 3 Running a Real Time Experiment Quick Guide After you click Run in the Workshop module the iQ5 software opens the Initiate Run tab within the Run Time Central module 2 3 1 Beginning a Run To begin a run 1 Insert the experimental plate into the iCycler reaction module Section 2 Quick Guides 2 From the Workshop Setup window select your desired Plate pts and Protocol tmo file individually or your desired Run Set run Click Run 4 Check that the desired Protocol and Plate Setup are displayed in the bottom half of the Initiate Run screen 5 Select the type of well factors to use by selecting either e Use Persistent Well Factors or e Collect Well Factors from Experimental Plate 6 Click Begin Run 7 Name the file with a unique name in the Save Optical Data File dialog box 8 Click OK 2 3 2 Monitoring the Run When the real time PCR detection system begins the run the iQ5 software opens the Monitor Run window Yo
78. alues to any newly added standards while Define is not selected you must open the Dilution Series dialog box again make any necessary edits and click Apply Dilution Series 4 2 9 Plate Setup Editor Spreadsheets There are two different spreadsheets available from the Plate Setup Editor window the Well Identifier and the Plate Setup Editor Spreadsheets Well Identifier Spreadsheet The Well Identifier spreadsheet Figure 4 10 opens beneath the representation of the experimental plate any time that the pointer tool is clicked on a well This spreadsheet simultaneously displays information about each dye layer in the active well In this spreadsheet you may edit the sample type identifier and quantity Any changes are applied to all members of a replicate group It is also possible to change the replicate group assignment of a well but doing so will also automatically change the assignment of all other members of the original replicate group NOTE In order to edit the replicate group assignment of a single well without changing the other members of the same replicate group make the change on the plate not in the spreadsheet Row Column Sample Type Rep Identifier Condition Quantity Units F 3 1 Beta Actin 1 00E 03 copy number 1 IL 1 Beta 1 00E 05 copy number Fig 4 10 The Well Identifier Spreadsheet Plate Setup Editor Spreadsheet The Plate Setup Editor spreadsheet can be opened by clicking the spreadsheet button in the Pla
79. ample Figure 4 9 Define Standards Replicates from f to 10 Starting Concentration e 00E 18 Series Identifier Bio Rad Dilution Factor fio 4 Increasing Decreasing Define Close Apply Dilution Series Fig 4 9 The Dilution Series Dialog Box 1 Access the dilution series box by clicking on Dilution Series 2 Define the range of standard replicates to be defined By default the beginning and ending standards are the lowest and highest numbered standards but you may define only a subset of all standards by clicking on the up or down arrows 3 Enter the starting concentration the concentration of the lowest numbered standard to be defined The Scientific Notation button controls only how the data are displayed in the spreadsheet 4 Enter a series identifier for the standard if desired It will be shown in the spreadsheet and in the reports 5 Enter the fold dilution factor The default value is 10 6 Indicate whether the concentrations decrease or increase as the standard replicates increase in number The default setting is Decreasing 7 Click Apply Dilution Series and all standard concentrations will be automatically calculated Adding Additional Standards When Define is selected at the bottom of the dialog box standards added to the set after the Dilution Series box is closed will have their concentrations automatically calculated 28 Section 4 Workshop Module To assign v
80. ample Gene Expressiontmo Selected Plate Setup Sample Gene Expressionpts Cycle 3 50X Sample Volume 25ul Seal Type Film Vessel Type Plates Step 1 Step2 95 ss 0 10 0 30 e a S Dwell PCR Melt Data Time Hold Setpoint Acquisition A o 95 0 C XM EST RU a erc TEC r E t Fig 5 2 The Initiate Run Window 5 2 Well Factors Well Factors are used to compensate for any system or pipeting non uniformity in order to optimize fluorescent data quality and analysis Well Factors may be collected directly from the experimental plate dynamic well factors or from an external well factor plate persistent well factors The best source of well factors for correcting non uniformity are dynamic well factors collected from the actual experimental plate However in order to collect well factors from the experimental plate the plate must meet certain requirements and be cycled for approximately 5 extra minutes 5 2 1 Dynamic Well Factors In order to collect dynamic well factors one of the following must be true e All wells must have the same combination of fluorophores AND the same concentration of fluorophores e No fluorophore is used both alone and in combination with other fluorophores AND no fluorophore is used in more than one combination of fluorophores The collection of dynamic well factors is a completely automated process which begins as soon as Begin Run is clicked and the f
81. an allele call the scatter plot reflects the change NOTE You may click and drag threshold bars directly on the plot to adjust Automatic Call assignment Click Reports in the menu to obtain customized reports for the allelic discrimination data 2 4 4 Gene Expression Quick Guide The iQ5 software can present expression data normalized to one or more reference genes or for data normalized before PCR as a relative quantity Calculating Relative Quantity dC AC To calculate Relative Quantity dCy In the PCR Quant screen with a opd data file open assess Threshold and Baseline information for the data file and make changes if necessary Click the Gene Expr tab 1 2 Make any changes to Gene and Condition for example Sample and Treatment assignments in the Gene Expression Plate interface Expand the Gene Expression Plate Interface view by clicking on the button to make well identification and selection easier Highlight the wells in the gene expression plate interface you wish to edit E c n Section 2 Quick Guides e Change the gene assignments in these wells by typing your desired name into the gene pull down men then click enter to apply the name to the selected wells e Change the condition assignments in these wells by typing your desired name into the condition pull down menu then click enter to apply the name to the selected wells NOTE The Gene and Condition Names have a character entry lim
82. and each of the five wells will contain an identical replicate number If there are not five wells left in the row the software will wrap around to the next row e Click a row letter the sample number of the first five wells will be incremented by one the next five wells incremented by two and the last two wells will be incremented by three The software does not automatically wrap around so that the last set of replicates has the same size as the others e Drag across a selection the first five members of the selection are incremented by one the second five members are incremented by two etc Numbering goes in a horizontal direction and wraps around to the next row within the selection The last set of replicates may have a smaller size than the others depending on the number of wells within the selected area With the Vertical direction column button active and the Size set at 3 if you e Click a well the sample type is incremented by one for the next three wells in the vertical direction If there are not three wells left in the column it will wrap around to the next column e Click a column number the sample number of the first three wells will be incremented by one the next three wells incremented by two and the last two wells will be incremented by three The software does not automatically wrap around so that the last set of replicates has the same size as the others e Drag across a selection the first three members of th
83. as plate e Optical quality sealing tape or preferred sealing method Before you perform background calibration complete the following steps 1 Prepare a background calibration plate see section 7 3 2 Ensure that the mask has been aligned 114 Section 7 Calibrating the Instrument 3 Place the background calibration plate in the iCycler thermal cycler 4 Select the Background tab in the Calibration module The Background window is shown in Figure 7 3 ra Mask EE Background NM WellFactors MMM Pure Dye Instructions Referto Help Fileforinstructions on howto run this type of calibration Seal Type Fim x Vessel Type Plates Collect Background Readings Fig 7 3 The Background Window To perform background calibration 1 Select a well sealing type film or caps from the drop down list 2 Select a vessel type plates or tubes from the drop down list 3 Click Collect Background Readings 4 When the iQ5 software completes the background calibration run a dialog box appears with the message Background Calibration Run Complete 5 Click OK to exit NOTE If you wish to calibrate for more than one vessel and sealing type combination repeat the process above to collect additional background calibration files During a run the software will automatically use the correct file for the vessel and sealing parameters specified in plate setup This completes
84. ate Setup Editor You can return to the Workshop at any time by pressing Cancel amp Exit Plate Editing Any current changes made since the file was last saved will be lost To save changes before returning to the home screen click Save amp Exit Plate Editing and assign a name to your new plate setup in a standard Windows Save dialog box All plate setup files are automatically assigned an extension of pts NOTE Plate setup files from earlier versions of the iQ5 iQ or MyiQ software can be opened iQ5 Optical System software version 2 1 but before they can be saved you must assign a vessel type and sealing mechanism if not previously defined 4 2 2 Well Definition Icons GJ E amp 2 S2 S X iX Fig 4 4 The Well Definition toolbar There are nine icon running across the top of the representation of the experimental plate Figure 4 4 These buttons are used to provide the two pieces of information required for each well sample type and fluorophore s to be monitored The active button is always surrounded by a red box Standard Pointer for selecting but not altering wells Defines Standards Defines Unknowns Defines No Template Controls oper 22 Bs ek Section 4 Workshop Module Defines Positive Controls Paint Bucket icon When the Paint Bucket icon is active and you click a well in the plate setup all wells in that replicate group will be assigned the currently selected fluorophore Delete Fluorop
85. atically exports the selected data into a protected workbook e The protected workbook generated by the iQ5 software contains the text values of what is represented on the spreadsheet For example checkboxes from the software application are replaced by True or False text in Excel e The numeric values contained in the protected workbook are exact values from the software application that include several non significant figures beyond the decimal point This is important to note when considering whether to transfer iQ5 data spreadsheets by a copy and paste command or the Export to Excel command With the copy and paste command only the significant digits displayed in the iQ5 software interface are transferred to Excel 6 5 5 Printing Results The Print command on the Results table menu will print the displayed spreadsheet Amplification Data or Standard Curve C Results When selected a Print Preview box is opened which contains an illustration of the spreadsheet as it will appear once printed Clicking the printer icon opens the Windows print dialog box Click OK to complete the printing task 6 6 Melt Curve and Melt Peak Charts The RFU data collected during the melt curve part of the experiment are plotted as a function of temperature as shown in Figure 6 22 The vertical temperature bar may be dragged to any position on the plot The temperature bar on the melt peak plot moves in sync to the same relative position on the melt curve
86. automatically 33 Section 4 Workshop Module Temperature Change You may program an automatic periodic increase or decrease in the step temperature in a repeated cycle Temperature increments or decrements may be as little as 0 1 C per cycle You may make the increase or decrease as frequently as every cycle and the increase or decrease can begin following any cycle The temperature increment or decrement may be as large as desired as long it does not result in temperatures which are outside the temperature limits of 4 100 C To program a temperature increment or decrement 1 Click Temperature Change in the Show Options box Three new columns will appear in the spreadsheet 2 For the repeated step you want to affect enter the incremental change desired in the Temperature change column To decrement the temperature enter the decremental change as a negative number for example 0 5 3 Enter the repeat in which you want the change to occur for the first time in the Begin Repeat column Usually it is cycle 2 but it can be any cycle greater than 1 4 Enter the frequency that you want the change to occur in the How Often column Usually you will want the change to occur every repeat so enter 1 in this column Time Change You may program an automatic periodic increase or decrease in the step dwell time in a repeated cycle Time increments or decrements may be as little as 1 sec per cycle You may make the increase o
87. ay be hot Allow at least 15 minutes after turning off the MyiQ2 MyiQ or iQ5 camera module before removing the lamp Lamp Replacement Procedure 1 Turn off the power to the optical module 2 Reach behind the optical module and unscrew the short fastener that secures the lid of the module in place 3 Using a gentle pressure with both hands push inward on the rear vents located on the top half of the instrument casing Lift upward to remove the cover of the optical module 4 The lamp is located on the right side of the optical module Push up on the lamp spring clip to release the lamp from the bracket 5 Lift the lamp out of the socket 6 Install the new lamp using the reverse of steps 1 5 Hold the new lamp by the outer reflector and do not touch the bulb Be sure the spring clip is down before inserting the lamp into the socket Push the lamp firmly into the bracket then close the case and secure the lid 135 Appendices Appendix A Warranty The MyiQ2 MyiQ and iQ5 Real Time PCR Detection Systems are warranted against defects in materials and workmanship For specific warranty information contact your local Bio Rad office If any defects should occur during the warranty period Bio Rad will replace the defective parts without charge However damage or defects resulting from any of the following causes are specifically excluded 1 2 3 4 5 6 Improper operation Use of improper solvent or sample Us
88. cells represented in each of your samples Relative quantity can be viewed as non normalized expression This value is sometimes referred to as dC or AC because of the equation used to calculate relative quantity By definition relative quantity data is not normalized Typically researchers that do not use reference genes are confident in one of the two following considerations Each condition sample assayed represents the same amount of biological sample Typically they choose to load the same mass of RNA or cDNA in each well and feel that mass of nucleic acid is an effective way of normalizing the resulting data No modification of relative quantity data is needed to obtain normalized data The data are normalized by experimental design Or Any variance in the amount of biological sample loaded will be normalized in post PCR analysis by some method For example a researcher might choose to simply divide the relative quantity value by the normalizing factor indicated after each of the examples listed below Options may include but are not limited to e Mass of nucleic acid loaded for each sample Rel Quant ng RNA represented in each sample 81 Section 6 Data Analysis Module e Number of cells from which nucleic acid was isolated Rel Quant number of cells represented in each sample e Mass of tissue from which nucleic acid was isolated Rel Quant mass of tissue represented in each sample To calculate Relative Quantity dC
89. click Make New Folder in the Browse For Folder dialog box A new folder will appear within the currently selected folder Figure 8 2 To rename this folder right click select Rename and then enter the new name of the folder in the text field next to the newly created folder Click OK when finished NOTE Changes to default File Paths DO NOT become active until the software has been restarted T Browse For Folder Boe Persistence ReportTemplates C3 RMEData SampleFiles SupportFiles z Be Users C3 admin 3 louise m gt a v l gt Fig 8 2 Creating New Folders in the Browse For Folder Dialog Box 123 Section 8 User Profiles 8 1 2 Plate Setup Preferences The Plate Setup preferences box Figure 8 3 can be used to define the default conditions when a user creates a new plate setup Plate Setup Concentration Units copy number v Seal Typedrim X Vesseld pistes SampleVolumej25 EA Scientific Notation DilutionFadorfio Dilution Series Increasing Decreasing Replicate Sze 1 EX Replicate Loading Row amp Column v Calibration Plates c Program Files Bio Rad jQ5 Users admin puredyestarte E Fluorophores v FAM Y SYBR v SYBRS Cy3 v SYBR1 v HEX TAMRA v SYBR2 TexasRe Y SYBR3 TET ROX Fig 8 3 Plate Setup Preferences The following default conditions can be set for the Plate Setup Concentration Units Choose fro
90. conditions displayed in the report template are different from the analysis conditions present at the time of the most recent file save event for a given dataset If this entry reads Yes then the displayed data was generated from an unsaved version of the dataset being analyzed e Notes Text entered in the Notes field at the time of a data file creation or data file save event is included in the header section of every report template e Protocol The header of each report template contains a summary of the protocol used for thermal cycling and data collection within a given dataset Any modifications to the protocol used for data collection will be displayed in the Report Header 6 12 3 Data Analysis Report Types There are over 15 different report templates available in the iQ5 software The number and type of reports available at any given time will vary according to the Data Analysis module currently being used The various report options available for each of the Data Analysis modules are summarized below PCR Quant Reports e PCR Quant Data This report template includes only the data included in the Standard Curve C Results spreadsheet the PCR amplification chart is NOT displayed 108 Section 6 Data Analysis Module e PCR Quant Detailed This report template includes all of the data available in the PCR Quant module of the iQ5 software including the PCR amplification chart Cr value spreadsheet and analysis parameters
91. d defaults to 2 for End Point Only runs and 5 for non End Point Only runs In order for the iQ5 software to analyze non End Point Only data in the End Point tab at least 6 repeats of data acquisition must be performed Number of Ranks The Number of Ranks allows assignment of samples into distinct groups based on their RFU values The absolute range highest RFU minus lowest RFU is divided by the number of ranks selected The default rank value is 10 and the minimum number of ranks is 3 Colored rank boxes displayed below Number of Ranks and Sort Data by Call symbolize the number and order of ranks in the end point analysis To the right of the colored rank boxes are five color gradation buttons that allow a change in the color scheme of the rank boxes once the data are analyzed Sort Data by Call This function is used to sort End Point samples into Positive and Negative Calls as well as No Calls wells which do not fall into the previous two categories Sort Data by Call is essentially the Positives amp Negatives method but also includes sorting and color coding the Positive and Negative Calls The ranking function is disabled in this mode er Section 6 Data Analysis Module Results The Results box lists the following information Source of Data Displays the analysis mode of the source data For End Point only data this is Background Subtracted For end point analysis of PCR quantification data the source of data must be e
92. d each experiment will be treated independently NOTE The software cannot distinguish between fluorophores read by the same filter pair in the same well therefore in all cases such dyes have to be in separate wells For example you may not put FAM and SYBR Green in the same well 24 Section 4 Workshop Module Fluorophore Probe Primer eios 3 sero i oses js oss sm i E Whole plate loading Select Add Fluorophores Fig 4 6 List of Five Sets of Wells Click Select Add Fluorophores to open the Fluor Selector box From this box choose up to five fluorophores to be monitored To remove a fluorophore from the list to be monitored uncheck the box next to its name To change the default color associated with a fluorophore click the color box on the row with the fluorophore name and choose a new color as shown below in Figure 4 7 Ma Bio Rad iQ5 Fluor Selector Fig 4 7 The Fluor Selector Window for the MyiQ2 Real Time PCR Detection System The iQ5 Optical System software automatically recognizes which instrument is detected and displays only the fluorophores appropriate to that system for selection Figure 4 7 illustrates the fluor selector window when connected to a MyiQ2 instrument You may add new dyes to the list at any time by clicking Add New Fluor When you add a new dye you must specify the filter to be used to
93. d extended to all replicates in the group NOTE To delete a previously defined well click the Delete All icon and then click the well To delete the selected fluorophore from a previously defined well click the Delete Fluorophore icon and then click the well 13 Click Save amp Exit Plate Editing The extension pts will be added automatically NOTE Selecting or creating plate setup files that contain fluorophores not compatible with the connected instrument will result in a warning message displayed at the bottom of the screen Figure 4 5 This message indicates that while the plate setup can be viewed and edited the plate setup contains fluorophores that cannot be used to collect new data using this plate setup AN Plate Setup is invalid Plate contains fluorophore s that cannot be used with the current instrument Fig 4 5 Invalid Plate Setup Error 4 2 4 Fluorophore Selection When using the MyiQ2 MyiQ and iQ5 systems you may specify as many as five different fluorophores on a single plate provided they are read through the filters available on the respective systems Refer to section 9 2 1 for information about system filter specifications and recommended fluorophores With the iQ5 software as many as five different sets of wells can be read with the same filter pair that is in the same fluorophore as shown in Figure 4 6 For example you may have five different experiments on the same plate all using SYBR Green an
94. de to any fluorophore within a well are extended to the other fluorophores within the well and within the replicate group If you are editing a plate the Whole Plate Loading checkbox may be unavailable because it is not appropriate based on the current definition of the plate 7 Click a fluorophore 8 Click a sample type icon 106 9 Section 6 Data Analysis Module Select the type of replicate loading desired 10 Click or drag across the plate to define wells with the selected fluorophore and sample 11 12 13 type Continue defining the remaining wells that will contain the first fluorophore by changing to any other sample type icons required To calculate standard concentrations automatically click Dilution Series and enter the upper or lower concentrations and units of the standards set the dilution factor and dick Apply Dilution Series Repeat steps 7 11 for any additional dye layers fluorophores as required Remember that if the Whole Plate Loading box is checked changes made in standard concentrations will be applied to all dye layers for that well and extended to all replicates in the group NOTE To delete a previously defined well click the Delete All icon and then click the well To delete the selected fluorophore from a previously defined well click the Delete Fluorophore icon and then click the well NOTE Use the Next checkbox to enter a particular number to assign to the next standard o
95. dient may be programmed across the reaction block at any step of a protocol The gradient runs from the back of the instrument to the front with the hottest temperature in row A and the coolest temperature in row H All wells in each respective row are at the same temperature so at any time during a gradient step there will be eight different temperatures across the block with 12 wells at each temperature The gradient may be as large as 25 C or as small as 1 C The gradient is not linear but is highly reproducible No row can be at a temperature higher than 100 C or lower than 40 C during the gradient step NOTE A gradient cannot be applied to any step which also has a melt curve Programming a Thermal Gradient To program a thermal gradient 1 Click Gradient in the Show Options box Two columns will appear in the spreadsheet and a representation of the gradient will appear on the right side of the window Figure 4 14 2 Click the Gradient checkbox in the spreadsheet for the desired step Dwell PCR Melt Data Time Setpoint Acquisition Gradient Range Cycle Repeats Step EN D 396 x E z 2 40 1 o 10 95 0 E 2 030 55 0 3 RealTime v 10 0 Fig 4 14 The Protocol Editing Table 3 The temperature listed in the Setpoint cell of the spreadsheet will be the coolest temperature on the block during the gradient step row H Enter the desired difference between the coolest and hottest temperatures during the gradient s
96. display the plate summary for a specific dye layer To print the selected plate setup display click Print To copy and paste the entire plate setup click Copy to Clipboard 30 Section 4 Workshop Module 4 3 Protocol Protocol files direct the operation of the iCycler thermal cycler and also specify when optical data will be collected during the thermal cycling run iQ5 software protocol files are stored with the extension tmo e A protocol is made up of as many as 9 cycles e A cycle is made up of as many as 9 steps e A step is defined by specifying a setpoint temperature and the dwell time at that temperature e Acycle is a collection of steps that are repeated up to 600 times Every protocol must have at least one data collection step This may be a real time data collection step or a melt curve data collection step Temperature and Dwell Time Ranges e Temperatures between 4 0 and 100 0 C may be entered for any set point temperature e Finite dwell times may be as short as 1 second 00 01 or as long as 99 minutes and 59 seconds 99 59 Minimum Dwell Times for Data Collection e 1 fluorophore 10 sec e 2 fluorophores 20 sec e 3 fluorophores 30sec e 4 fluorophores 40 sec Bio Rad recommends using a slightly higher dwell time than the minimum values so that more data points are collected at each repeat 4 3 1 Selecting a Protocol 1 With the Protocol tab active use the browser to locate the folder where t
97. during system operation Do not attempt to remove the lamp without powering off the instrument and allowing the system to cool for at least 15 minutes To prevent skin burns and fire hazards do not attempt to operate the real time PCR detection system while the camera case is open Do not open casing of the optics module when the instrument is in use Operating Temperature For normal operation the maximum ambient temperature should not exceed 40 C To ensure adequate cooling of the system maintain a clearance of at least 4 inches around the sides of the MyiQ2 MyiQ or iQ5 optics module Do not block the fan vents near the lamp as this may lead to improper operation or cause physical damage to the detector Do not operate the optics module in extreme humidity that is greater than 90 percent or where condensation can short internal electrical circuits or fog optical elements Notice The MyiQ2 MyiQ and iQ5 instruments are designed and certified to meet EN 61010 safety standards EN 61010 certified products are safe to use when operated in accordance with the instruction manual These instruments should not be modified in any way Alteration of the instruments will e Void the manufacturer s warranty e Void the EN 61010 safety certification e Create a potential safety hazard Bio Rad is not responsible for any injury or damage caused by the use of these instrument for purposes other than those for which they are intended or by modifica
98. e The options are Positive Negative or blank Alternatively you may type the letter p or the plus sign to select a positive control and you may also type the letter n or the minus sign to select a negative control The controls specified in this column are used in the end point analysis calculations that assign positive or negative values to the unknowns You may click on the Define Controls column header to sort the column by the controls e Unknowns Call If a well is not defined as a positive control or a negative control in the Define Controls column it is considered an unknown for end point analysis This column displays the value assigned to each unknown after Recalculate has been clicked Unknowns Call may be Positive Negative or blank You may click on the Unknowns Call column header to sort the table by Unknowns Call e Unknowns Ranking Lists the rank into which each unknown falls An unknowns ranking depends on the total number of ranks and the end RFUs value You may click on the Unknowns Ranking column header to sort the table by Unknowns Ranking e Identifier Lists the identifier for every well as defined in the plate setup You may click on the Identifier column header to sort the table by identifier 6 8 3 Recalculate Button Clicking Recalculate performs the final calculations used to determine unknown sample calls The Recalculate button is active when sufficient controls have been specif
99. e Expression Analysis 1 In the PCR Quant screen with a opd data file open assess threshold and baseline information for the data file and make changes if necessary 2 Click the Gene Expr tab 3 Make any changes to the Gene and Condition for example Sample and Treatment assignments in the gene expression plate interface 4 Set reference genes if required and choose an analysis method e Normalized Expression ddC is the default or e Relative Quantity dC 6 OPTIONAL Assign attributes sample color show graph etc and user defined reaction efficiencies in the Gene List 7 OPTIONAL Assign attributes sample color show graph etc and set control samples in the Condition List 8 Click Recalculate to see your results If you want to compare these data to results obtained in other opd files you will need to enable this file for Multi file Gene Expression analysis also called a Gene Study To enable your file for Gene Study 1 Click Enable for Gene Study 2 Goto the File menu to save your file Normalized expression is graphed in the gene expression graph interface The data can be accessed in the Gene Expression Settings interface within the Data Table 6 9 1 Normalized Gene Expression ddC AAC Rather than using some other method to normalize data you may use the measured expression level of one or more reference genes as a normalization factor Reference genes should be genes which are not regulated
100. e displayed above Increase exposure time and collect a new dien if the fluorescent signal eu the loaded wells is too low a Confirm that small green boxes are visible around each well where sample fluorescence will need to be detected 3 Select the next fluor on the pause ion list and repeat steps 1 and 2 for all additional dyes in the experi When finished return tothe Initia Tnitiate Ru ae screen Fig 5 4 The Show Plate Window The following steps describe typical use of the Show Plate window 1 Collect an exposure from the camera by clicking on Expose An image of the fluorescence from the sample block area will be displayed NOTE If the image displayed is too bright or too dim it may be necessary to adjust the exposure time using the pull down box in the right hand corner of the Show Plate window 2 Confirm that the small green boxes are visible around each well where sample fluorescence will need to be detected If necessary open the instrument and correct sample positions to match the plate setup for sample detection 44 Section 5 Run Time Central Module 3 Ifa multiplex assay is being performed repeat steps 1 amp 2 for all fluors present in Fluor Selection list 4 When finished return to the Initiate Run screen 5 6 Monitor Run Window Open the Monitor Run window by clicking the Monitor Run tab The window appears as shown in Figure 5 5 Traces are displayed in real time and run prog
101. e files see Gene Study EXE cen ENH Data Table Gene List Condition List Data Set List Name Full Name Created Date B E SYBR Timecoursel opd 1 11 2008 1 51 18 PI 2 2 SYBR SYBR Timecourse2 opd 1 11 2008 3 39 44 PI Fig 6 45 Accessing and viewing the Data Set List 88 Section 6 Data Analysis Module 6 9 5 Applying an Analysis Method 1 Choose an analysis method by clicking one of the option buttons e Normalized expression ddCt is the default and requires you to first choose a reference gene or e Relative quantity dCt 2 Click on Recalculate to apply the new settings NOTE Many changes made to the plate setup and analysis options require that you recalculate If you do not immediately see the change you expect to see click Recalculate Figure 6 46 Normalized expression ddct Relative quantity dc Fig 6 46 Applying an Analysis Method 6 9 6 Data Table for Gene Expression Analysis The Data Table spreadsheet for gene expression analysis is accessed by clicking Data Table The Data Table spreadsheet lists the Condition and Gene names and calculated expression and C values Figure 6 47 Columns can be resized and re ordered by clicking and dragging Any condition being used as a control is indicated by an asterisk in the Ctrl column Columns can be sorted by Condition or Gene name by clicking on the sort triangle icon next to the column heading qum Corrected Conditi
102. e mouse cursor is positioned on the chart data bars the resulting Tool Tip will display the data label that is Gene or Condition Name depending on which x axis mode is selected along with the calculated Expression value in parenthesis When the mouse cursor is positioned on the chart error bars the resulting Tool Tip will display the Condition Error or Gene Error depending on which x axis mode is selected The error value displayed is the Unscaled Expression standard deviation However the Tool Tip will display the Corrected Unscaled Expression standard deviation if Corrected Std Devs is selected from the Gene Expression context menu Copy Graph Export a Graph To export a graph 1 Place the mouse pointer over the graph 2 Right click on the graph A shortcut menu appears 95 Section 6 Data Analysis Module 3 Click Copy Graph 4 Switch to or open the document into which you will paste the graph 5 Paste the graph image by choosing Edit then Paste or by pressing CTRL V 6 9 8 Normalized Expression Calculations The normalized expression calculation is stated below To see how the iQ5 software calculates relative quantities go to relative quantity calculations Relative Quantity ampie ene Normalized Expression sampe gene x Rel Quant Rel Quant sampe 45 Rel Quant sampe petm sample Ret 1 Normalized Expression when a Control is Chosen When a control is chosen the iQ5 software uses the e
103. e selection are incremented by one the second three members are incremented by two etc Numbering goes in a vertical direction and wraps around to the next column within the selection The last set of replicates may have a smaller size than the others depending on the number of wells within the selected area The Next box allows you to overwrite the default replicate number for the next sample to be defined This is an easy way to correct a mistakenly assigned replicate number Use the up and down arrows to select the next desired replicate number or highlight the field and type the desired number 4 2 7 Sample Volume Seal Type and Vessel Type In Plate Setup you must specify the sample volume the type of sealing and the type of vessel to be used in the experiment This is to properly associate the appropriate calibration files unique for each vessel and sealing combination with the data file Use the up and down arrow keys to specify the sample volume or highlight the field and type in the volume Select the seal type and vessel type from the pull down menus 27 Section 4 Workshop Module Sample Volume 5 Seal Type Film Vessel Type Plates m Fig 4 8 The Seal and Vessel Type Dialog Box 4 2 8 Defining a Dilution Series The iQ5 software can automatically calculate the concentrations of each member of a dilution series given the dilution factor and the concentration of either the most or least concentrated s
104. e wide polymorphism screening Nature Genetics 9 341 342 1995 6 7 10 Restore Default At any time all modifications to the allelic discrimination data can be reversed by clicking Restore Default The iQ5 software will reload the original x axis allelic 1 fluorophore and y axis allelic 2 fluorophore data original display mode and Show Labels setting that were originally saved in the opd file and will also set the Automatic Call method to recalculate the data plot the chart and display the results in the allelic discrimination data spreadsheet 6 8 End Point Analysis The End Point analysis module is a convenient method of analyzing final relative fluorescence unit RFU values End point analysis can be performed in two ways e Selecting the End Point tab in the Data Analysis module for an existing data file e Click Run End Point to initiate the collection of End Point data from a sample plate Newly collected End Point data is displayed immediately following an End Point run Any existing opd data file can also be viewed in the end point module however the corresponding PCR quantification data must be analyzed in either the PCR Base Line Subtracted Mode or the PCR Base Line Subtracted Curve Fit mode before the End Point tab becomes available Although any file with amplification data may be analyzed post run in both the PCR Quant and End Point tabs an End Point run may only be analyzed in the End Point tab Melt Curve only experiments
105. e with tubes plates or sealing materials not specified by Bio Rad Laboratories for use with the MyiQ2 MyiQ or iQ5 Real Time PCR Detection System Deliberate or accidental misuse Repair or modifications done by anyone other than Bio Rad Laboratories Natural disaster of any kind The warranty does not apply to fuses For inquiry or request for repair service contact Bio Rad Laboratories after confirming the model and serial number of your instrument For technical support call your local Bio Rad office or in the US call 1 800 4BIORAD 1 800 424 6723 or visit us on the Web at discover bio rad com 136 Appendices Appendix B Troubleshooting Error Messages A Plate Setup is invalid Plate contains fuorophore s that cannot be used with the current instrument Cause Selecting or creating plate setup files that contain fluorophores not compatible with the connected instrument The warning message above will be displayed at the bottom of the screen This message indicates that while the plate setup can be viewed and edited the plate setup contains fluorophores that cannot be used to collect new data Solution If you wish to view or edit the plate setup no corrective action is required If you want to run this plate setup using the currently connected instrument you will need to edit the plate setup to remove fluorophores that are incompatible with the connected instrument Refer to section 4 2 for further details A Dat
106. eT Ee poe Vue 74 6 7 8 Horizontal Threshold iere nnn nnn a nh nh ah a nk Hk a Ta genka aa due 74 6 79 Normalize Data eiit ek erbe eiua tha ERR a D FEE Rea LE La aaa a TR CR ERE RERE E 74 6 7 10 Restore Default idera rere e rna aR AAA NAAA T A KRNRERERRR CHER ERA RR EREKL CERA RR Nadana 75 6 8 End Point Analysis neos Loi ree xa ee oc ern eure as CE a ra ee ER xe E CEDE RR E ERR UE 75 6 8 1 The End Point Analysis Settings cccccceecceeeeceeeeeeeeeeeeseeseeeseeeeaeseeesaeseeeseseeeesieeeaeeea 75 6 8 2 End Point Analysis Spreadsheet sese nennen nennen nnns 77 6 8 3 Recalculate BUttOn iini tenete Dur FRE a ewe tere LE Da hee ERE a EEAO 78 6 9 Gene Expression Analysis 2 ici erieie ve cioe edere ese roe spa te revu ak vedo gra vg oe ODIO Pa A Xa Rode 78 6 9 1 Normalized Gene Expression ddCr AAC een 80 6 9 2 Relative Quantity QC ACy uia aiiarspecac eria pne Doer ID RO CHO E A GE X TD 81 6 9 3 Specifying Gene and Condition Labels for Gene Expression Analysis 82 6 9 4 Setting Analysis Parameters for Gene Expression Analysis Using the Settings Tab 85 6 9 4 1 Gene List Assigning Reference Genes and Target Specific Reaction Efficiency 85 6 9 4 2 Assigning and Naming Control Samples Using the Condition List 87 6 9 4 3 Data Set EISE de eec ne d ete e ee f ete 88 6 9 5 Applying an Analysis Method
107. eans that you are doubling your sequence of interest with each cycle People focus on efficiency for a number of reasons There is evidence that using accurate efficiencies for each of your gene primer probe pairs will give you more accurate results when using the mathematical modules used in the Gene Expr tab You set the efficiency for each of your genes in the Gene List The default value is 100 So if no standards were included on the plate by default an efficiency value of 100 will be reported This efficiency value is fully editable to allow entry of other than a default 100 value established from previous experiments If a standard curve was included on the plate the software will automatically calculate the efficiency for that gene and report it in the Efficiency column To use Auto Efficiency you must have standards in your experiment that result in valid standard curves in the PCR Quant tab The iQ5 software only requires two standards at different concentrations though it is recommended to have at least four samples in triplicate across a relevant dynamic range 6 9 4 2 Assigning and Naming Control Samples Using the Condition List When the Settings button is active and Condition List is selected the conditions being analyzed in the experiment are listed along with their full name This Full Name column is editable so that longer more descriptive names may be entered if your Condition Name entry was abbreviated NOTE Conditi
108. elect the desired data setup file from the file tree directory Click the file name to open the plate setup associated with the data file in the bottom right section of the Workshop window Click Edit to open the plate setup in the Plate Setup Editor Enter or edit any notes about the plate setup in the Notes box Enter or edit the sample volume seal type and vessel type Enter or edit a name for the experiment wk Ww N Click Select Add Fluorophores and select or edit the fluorophores to be used on the plate 6 For most experiments leave the Whole Plate Loading box checked With Whole Plate Loading changes made to any fluorophore within a well are extended to the other fluorophores within the well and the replicate group If you are editing a plate the Whole Plate Loading checkbox may be unavailable because it is not appropriate based on the current definition of the plate Click a sample type icon Select the type of replicate loading desired Click a fluorophore 10 Click or drag across the plate to define wells with the selected fluorophore and sample type 11 Continue defining the remaining wells that will contain the first fluorophore by changing to any other sample type icons required To calculate standard concentrations automatically click Dilution Series and enter the upper or lower concentrations and units of the standards set the dilution factor and click Apply Dilution Series 12 Repeat steps 7 11 for any
109. er Click the protocol name once The selected protocol appears in the Selected Protocol pane NOTE The iQ5 software has a number of sample protocol files which may be used Section 2 Quick Guides Editing or Creating a Protocol Edit the selected protocol by clicking Edit in the Selected Protocol window or create a protocol from a protocol template by clicking Create New in the Selected Protocol window NOTE Clicking Edit or Create New in the Selected Protocol pane opens the Protocol Editor NOTE You can only exit the Protocol Editor by clicking Save amp Exit Protocol Editing or Cancel amp Exit Protocol Editing 1 Edit the protocol by performing one or more of the following five tasks Edit the Dwell Time and Setpoint temperature Click in the Dwell Time or Setpoint cell and then enter the Dwell Time or Setpoint temperature To enter 10 seconds type 0 followed by a colon then type 10 that is as the time appears in the spreadsheet Alternatively 10 seconds can be entered as 0 10 Set Data Acquisition Step Click in the Data Acquisition column and then click Real Time at the step you want to collect real time data Click Melt Curve if data from a melt curve is required NOTE Ensure that every thermal protocol has at least one Data Acquisition step Insert a cycle Insert a cycle by clicking in the Insert column within the cycle row Cycles have a blue background The iQ5 software inserts the new cycle below the cu
110. er to apply the name to the selected wells 15 Section 2 Quick Guides e Change the condition assignments in these wells by typing your desired name into the condition pull down menu then click enter to apply the name to the selected wells NOTE The Gene and Condition Names have a character entry limit of 15 characters A n e Minimize the Gene Expression Plate interface by clicking on the button to return to the standard view of the Gene Expr tab window 7 Select either Normalized expression ddCt or Relative quantity dCt NOTE For Normalized Expression analysis you must first assign reference gene s Click the Settings tab then select Gene List spreadsheet to set your desired reference gene s Click Recalculate to see your results Normalized Expression results are graphed The Data Table spreadsheet is accessed by clicking Data Table The Data Table spreadsheet lists the Condition and Gene name calculated expression values and C values Right click on the spreadsheet to print or export this data to Excel NOTE Click the Settings tab then select Gene List to enter a specific user defined gene reaction efficiency NOTE Use the Settings tab then select Condition List to select a particular sample as a control sample 2 4 5 Post Run Plate Setup Editing Quick Guide For post run editing of the Plate Setup saved within a data file 1 From the Workshop module e Click Data File above the directory of the home wor
111. ere are two intra cycle data filtering options available Rolling Boxcar and Weighted Mean The default filter is the weighted mean as this filter is the only one available during data acquisition Intra Cycle Rolling Boxcar Weighted Mean Disable All Enable Global Filter OK Cancel Fig 6 14 The Set Digital Filters Window The weighted mean is determined by the equation O R c M 1 c 58 Section 6 Data Analysis Module Where e Oiisthe filtered value for a given data point i eR is the unfiltered value for data point i e cis a weighted factor with a value of 2 e Mis the arithmetic mean of all data points for the well within the given cycle The rolling boxcar filter is the arithmetic mean of the data readings i w 1 where w is the filter width For example if you want to calculate the 20th data point i220 and the width is 4 w 4 then you take the mean of the data points 17 20 data point 21 is the mean of the data points 18 21 etc The rolling boxcar filters only apply within a cycle A global filter that smoothes data from cycle to cycle is also available by clicking Enable Global Filter The global filter operates on the trace for a given well and fluorophore using all cycles together in a single pass Global filtering should be reserved for data that appears very noisy and should not be applied routinely Base Line Threshold Settings In the amplification chart the iQ5 software uses the
112. ernate After setting to Never c For USB Settings set USB Selective Suspend setting to Disabled d Set Search and Indexing Power Savings Mode setting to Balanced e Set Display Turn Display Off After setting to Never When completed click Apply and then Done Computer Power Management Settings on Windows XP Ur cBe gal Tes o 10 11 From the Start menu choose Settings then Control Panel Choose Power Options the Power Options Properties window will open On the Power Schemes tab set all power scheme settings to Never On the Hibernate tab deselect the Enable Hibernation checkbox Return to the Control Panel window and select Display the Display Properties window will open On the Screen Saver tab select None Return to the Control Panel window and select System the System Properties window will open On the Hardware tab click on the Device Manager button to open the Device Manager file tree Click on the button to expand the Universal Serial Bus Controllers list Scroll down and highlight the first listed USB Root Hub Right click and select the Properties option to open the USB Root Hub Properties window Select the Power Management tab and deselect the Allow the Computer to Turn Off This Device to Save Power checkbox Repeat steps 9 10 for each individual USB root hub listed Section 1 Getting Started 1 5 Calibrating the Instrument Before using the real time PCR detect
113. esseeeeeeeeeeeee nnnm nnn nnn nnns 89 6 9 6 Data Table for Gene Expression Analysis sese 89 6 9 7 Graphing Options for Expression Data eeeseeeeeeeeeeeee nmm nnns 92 6 9 8 Normalized Expression Calculations eeeeseeeeeeeeeeeeeeee nemen nnns 96 6 9 9 Relative Quantity Calculations eese enne nennen nnne nnn nnns 97 6 9 10 Gene Expression Frequently Asked Questions eeeeeeeeeeeeennm enne 98 6 10 Gene Study Multi File Gene Expression Analysis eeeeeeeeeeenn nnn 99 6 10 t The Gene Study File ioter eco oe ett dn eo eek teer Ee sedi vue a eU edge das 99 6 10 2 Creating a Gene Study File esseseeeseeeeennenenenenenenenenennnennnen nnns 101 6 10 3 Gene Expression Study Manager eeeeseeee nennen nennen nennen nnns 102 6 1 0 4 Editing a Gene Study iei gerer re tb nae Ey R RR PER ARERERFRRER DR deehcaceta necededeanedweass 102 6 10 5 Excluding Including Samples Using the Gene Expression Plate Interface 103 6 10 6 Inter Run Calibration svcccccccicecescsvccccesaccececcavececacdiecdeaedvcedeacadecceccaveccncvavecdeacvaecceaeds 103 6 11 Post Run Plate Editing rte ted ihn anh unn kia k a dectebecdeetiodcecanceeueecuideddeess 105 Ju PAR Pe s oro ue TOO e M 107 6 12 1 Report Viewer Options ie ngrira riera raanei raia a eene nnne nnne nn nnne nn nnne nnn nn nnne 108 6 12 2
114. esser The highest expresser for each gene has a value of 1 e Scale to Lowest This option recalculates the normalized expression for each gene by dividing the expression level of each condition by the lowest expresser The lowest expresser for each gene has a value of 1 e Unscaled This option does not scale to any sample in particular It presents unscaled normalized expression e Scale to Control Scale to control is another scaling option which is accomplished by assigning a control in the Condition List x Axis Options You may graph either genes or conditions on the x axis by changing these options Figure 6 53 Graph 0ata 3 E BR Dus O nelativeto cortr Relative to zero 9 140 01 g M i 5 P 1 0060 3 Y iw Scaling Options 3 92 a ae eiu i Quse vem LEO Y Tulio i S Devs Fig 6 53 Selecting x axis Grouping Options 93 Section 6 Data Analysis Module Graph Error Options The default presentation for the error bars is one standard deviation Figure 6 54 You can change the multiplier to get 2 or 3 standard deviations E Grach Date LM I ORneistivet x Axis Options 34 Condton e yds Opbons IE Over Sang Opbers eon e ursaied Nor makrod Fold E xprensaon o w o E n t amp t u pg ia s in 2 2 2 d 2 amp U 9 RR R momo m m m 7 E 2 8 8 2 gz E4 4
115. et are approximately equal 49 Section 6 Data Analysis Module Use the fluorophore selector buttons to select and deselect which fluorophores to display De selecting a fluorophore removes it from the display but not from the analysis selected fluorophore buttons are displayed with a red border around their perimeter De selected fluorophores are displayed without this red border In Figure 6 3 the FAM fluorophore has been selected and HEX has been de selected on ea Fig 6 3 Fluorophore Selector Buttons 6 2 2 Select Analysis Mode You can select from three options in the Analysis Mode drop down list box Figure 6 4 Analysis Mode PCR Base Line Subtracted Curve Fit Y Background Subtracted PCR Base Line Subtracted PCR Base Line Subtracted Curve Fit Fig 6 4 The Analysis Mode Drop Down List The three analysis modes include Background Subtracted The background subtracted data is the relative fluorescence of each fluorophore after normalizing for exposure time background factors and well factors No further analysis is possible on background subtracted data therefore the End Point Allelic Disc and Gene Expr tabs are unavailable PCR Base Line Subtracted To determine threshold cycles construct standard curves and determine the concentration of unknown samples the data must be PCR baseline subtracted The iQ5 software determines each PCR baseline subtracted trace by fitting the best straight line thro
116. et and enter a name for your new run set 4 5 Data File This feature is primarily used to select and open a data file It can also be used to run a real time experiment using the same protocol and plate setups that were used to create the data file The plate setup that is selected can be either the Original or Current last saved plate setup 38 Section 4 Workshop Module 4 5 1 Selecting a Data File 1 Click the Data File button 2 Use the file browser below the Data File button to locate the folder that contains the data file and then click on the file name to select it 3 The protocol and plate setup used in the data file will appear in the Selected Protocol and Selected Plate Setup windows The plate setup that is shown can be either the Original or Current last saved plate setup If the data file protocol and plate setup are being used as the conditions for running a new experiment then the plate setup that is shown in Selected Plate Setup window will be the one that is used to run the new experiment 4 5 2 Opening a Data File To open an optical data file from an amplification or melt curve experiment select the name of the data file in the file browser and click Analyze or double click the file name NOTE Data files collected using an iQ5 Real Time PCR Detection System can be opened when the computer running iQ5 software version 2 1 is connected to a MyiQ2 or MyiQ Real Time Detection System A warning message will
117. experimental goal may be to analyze samples conditions over a fixed time course In both cases it is essential to perform gene expression analysis on data generated in different data sets To accommodate this need a Gene Study file can be created in the iQ5 software 6 10 1 The Gene Study File A Gene Study file is a specialized file consisting of data and sample information imported from multiple opd files The imported data is grouped into a single study which can be edited files added or removed and further analyzed at the users discretion Gene Study files are assigned a file extension of gxd by the iQ5 software A Gene Study file is capable of comparing approximately 5 000 total wells of data This implies a maximum comparison of approximately 52 opd files containing 96 wells of single fluor non multiplexed data or 10 opd files containing 96 wells of five fluor multiplexed data Many other combinations of file number well capacity and fluor number are possible The absolute well maximum will depend on the amount of RAM and virtual memory available to your computer Enable for Gene Study Button All opd files must be enabled for gene study analysis from within the Data Analysis module of the iQ5 software Enabling an opd file for gene study analysis allows the iQ5 software to extract only the information critical to gene expression analysis between different files This step is 99 Section 6 Data Analysis Module perfor
118. f the C of the sample Relative Quantity sample Relative Quantity of sample E Efficiency of primer primer probe set this efficiency is calculated as follows Efficiency 0 01 1 where 100 2 6 9 10 Gene Expression Frequently Asked Questions Why should I normalize my data Relative quantity data that is not normalized by some means is difficult to interpret Imagine the case where you load 1 ug of RNA in one well and 10 ng in the other well If you perform a relative quantity analysis on the results from such an assay the fact that the 10 ng sample has a smaller relative quantity value is irrelevant It is likely the result of using less RNA and not the result of some biological response How does normalized expression calculated by this software compare to the value calculated using the ddC equation If you leave efficiencies at 100 and only evaluate one reference gene and one gene of interest the software will generate the same results as you would get using the ddC equation The standard deviations will be larger since the error propagation outlined in the initial publication is inappropriate 98 Section 6 Data Analysis Module How does normalized expression as calculated by this software compare to the model introduced by Dr M Pfaffl et al If you only evaluate one reference gene and one gene of interest you will get exactly the same results using the iQ5 software as you would using the model introduced in t
119. f the Setup window the Selected Data File area displays the selected data file or the run set file name that you selected in the file browser area The Notes box displays the notes associated with either the run set or data file Buttons in this area include e Run Used to initiate a real time PCR experimental run e Begin End Point Used to initiate an End Point run e Analyze Used to open a data file You can also open the data file by double clicking on the data file name in the file browser area 4 2 Plate Setup The MyiQ2 MyiQ and iQ5 Real Time PCR Detection systems only display and analyze data from wells defined in plate setup as containing sample and at least one fluorophore In the Plate Setup Editor window you specify the type of sample and the fluorophores present in each well The Plate Setup Editor window is accessed from the main Workshop module window by clicking on either Edit or Create New in the Selected Plate Setup window see Figure 4 1 or by double dicking on an existing plate setup file from the file browser folder tree 4 2 1 Plate Setup Editor Window The Plate Setup Editor window Figure 4 3 is comprised of a 96 well plate layout functions for specifying the sample type and fluorophores in each well and a spreadsheet displaying the definition in each fluorophore for any individual well 21 Section 4 Workshop Module Fig 4 3 The Plate Setup Editor Window Exiting the Pl
120. fold induction of one gene relative to another gene or relative to itself under different circumstances for example temporally geographically or developmentally different points e Edit Plate In the Edit Plate screen you may make changes to the sample type assignment or to the quantities of the Standards in the plate setup used to run the experiment This is a simple way to salvage your experiment should you make mistakes in the sample type assignment While you may make changes to the plate setup the original plate setup is never discarded and always remains with the data file so that you may revert to it at any time 6 1 PCR Quant Tab The PCR Quant tab is the tab that is first displayed after opening a data file that contains amplification data Figure 6 1 For experiments that lack amplification data such as Melt Curve only or End Point Only experiments the PCR Quant tab is grayed out and unavailable for analysis Use the PCR Quant tab to set the analysis conditions for the data file The analysis conditions include setting the PCR baseline setting the threshold and determining which wells to exclude or include in the experiment The analysis conditions should be set before using the Gene Expression End Point or Allelic Discrimination tabs For experiments with standards of known quantities the PCR Quant tab is also where you can determine absolute quantities for unknown samples You can also determine the efficiency of the PCR re
121. ge eap Rua ea eu ka a ek a agua a a ga ped Eua ad 20 4 1 2 Selected Protocol Area ieiiie eiie ree ianuae nane urhe Re RE ELE Rag Lea SEVERE Enna PEEL gau aas 20 4 1 3 Selected Plate Setup Area eeeeeeseeseeeeeeeee nennen enne nnne n nnn nnn s nnne nnn nnn nnn 20 41 4 Selected Data File Area aise telsiceteccdeed eens Ree ke a a i Ed E eve Ec Ya TE oira 21 4 2 Plate Set p 5 eer UI eere eed DeC Pd De ca coe Fe i done edes E s 21 4 2 1 Plate Setup Editor WindOw cccccccsstseeeeeeeeeeseae sneer teense nnne nnne nnn nnn nnn nnn nnne 21 4 2 2 Well Definition ICONS c ecsccscnesedeeeceaneateadiceexencaesananedetsed scans decececseusacasssancedeasedqeatedanad 22 4 2 3 Editing or Creating a New Plate Setup ccccccceceeecececeeececeeeeeeeseceeesesesanananeseenenes 23 4 2 4 Fluorophore Selection oo eer renner eee nnn nn nnn nnn nnn nnn 24 4 2 5 Whole Plate Loadihg rena ada aa kh aaa eua agen ka FER ua a 26 4 2 6 Specdifying Replicates 5 eet eot SEO a de a ESO re greed ace 26 4 2 7 Sample Volume Seal Type and Vessel Type eeeeeeeeeeenenennnne nnne 27 4 2 8 Defining a Dilution Series suueeeeeeeseseeeeeeeseseeeeeeenenenennnnnnnnnnnnnnnnnennn nnne nnns 28 4 2 9 Plate Setup Editor Spreadsheets seeeeeseesseeeeeee nennen nnnm nnn nnne 29 42 10 Plate S mtmnaty eic e m eene cent a o E arene ai ceo eve eode eva A 30 ecu
122. he following steps 1 Prepare an external well factor plate see section 7 3 2 Place the external well factor plate in the iCycler thermal cycler 3 Select the Mask tab in the Calibration module to open the image window Figure 7 1 ES Mask Background Well Factors Pure Dye Filter Position Image Adjust Mask IU Cres as o Left Right Down e Optimize Mask _ Ell Show Mask BB Wr correction Home Save Mask Exposure Time ms Reload Mask 2048 Restore Factory Mask Take an Exposure Well Camera Status Serial Number EP0021 Background p FW version 1 032 Fluorescence XR rerage Innen Library version 1 026 Fluorescence Model IQ2COLOR Average Outer FECE Fluorescence Lamp Time tasted pe Warmup Time Fig 7 1 The Mask Image Window for the MyiQ2 Real Time PCR Detection System The main use of the Mask Image window is to adjust the masks although there are other uses including capturing an image of the experimental plate to check the response of a probe or to assess the completion of a reaction With a plate image you can obtain fluorescence readings for each of the 96 individual wells To align the mask 1 Click Home 2 Click the Filter Position 2 option button 113 Section 7 Calibrating the Instrument 3 Click Take an Exposure The Image screen displays the fluorescence detected by the CCD camera for each of the 96 wells on the plate Pixels
123. he protocol is located and then click on the protocol name to select it The selected protocol appears in the Selected Protocol pane Figure 4 13 3 The iQ5 software has a number of sample protocol files you can use 23 Section 4 Workshop Module C RepotT engi StopampeMet tmo RMEData 2stepatandard tmo SupotFie r Gradert wo C User MelCurve tmo welfacon g Q Mo Pa 51 1591n 95 0 60 0 Q9 Rea Time Fig 4 13 The Protocol Selected Protocol Pane 4 3 2 Editing or Creating a Protocol To edit a selected protocol double click the protocol file or click Edit in the Selected Protocol window To create a protocol from a protocol template click Create in the Selected Protocol window Clicking Edit or Create in the Viewing Protocol window will transfer the software to the Protocol Editor You can edit the protocol in the spreadsheet at the bottom of the Editing Protocol window e Editing Dwell Time and Setpoint Temperature Click in the spreadsheet in the Dwell Time or Setpoint cell then enter the dwell time or setpoint temperature e Data Acquisition Click in the spreadsheet in the Data Acquisition column and select real time data collection for any step Choose Melt Curve if data from a melt curve is required NOTE Ensure that every thermal protocol has at least one data acquisition step e Inserting Cycles and Steps To insert a cycle click in the Insert column on the Cycle row C
124. he two strands of DNA are annealed As the temperature is raised towards the Tm of the duplex the fluorescence will decrease at a constant rate constant slope At the Tm there is a dramatic reduction in the fluorescence with a noticeable change in slope The rate of this change is determined by plotting the negative first derivative dF dT versus temperature The greatest rate changes yield visible peaks representing the T of the dsDNA complexes A melt curve cycle may follow an amplification cycle or can be conducted independently of the amplification The melt curve may be programmed in the following manner 1 Melt curves are programmed as a repeated cycle containing only one step The temperature is programmed to increase or decrease incrementally with each repeat of the cycle The increase or decrease combined with the number of repeats may not result in a temperature that is below 4 C or above 100 C at any time during the protocol 2 Insert a cycle into the protocol at the point that you want the melt curve The step generated in this cycle will be used to generate the melt curve data 3 Enter the temperature at which you wish to begin the melt curve in the Setpoint cell 4 1009C 4 Enter an appropriate dwell time for data collection under the Dwell Time column Dwell times for the melt curve will vary based on the number of fluorophores in the experiment To enter 10 seconds type 0 followed by a colon then type 10 that
125. he type of sample and fluorophore simultaneously one dye layer at a time e When Whole Plate Loading is toggled off changes made to one fluorophore are considered unique to the fluorophore and are not automatically extended to the other fluorophores in the well However these changes are extended in the same fluorophore to every other well within the replicate group 4 2 6 Specifying Replicates The buttons for specifying replicates run down the left side of the representation of the experimental plate 1 This is the default setting and indicates that each sample is identical that is a single replicate With this button active if you rere e Click an unused well the sample type is incremented by one e Click a row letter or column number the sample type for every member of the row or column is assigned the identical replicate number e Drag across a selection the sample type for every member within the selection is assigned the identical replicate number 26 123 D QN eu Size il 123 e a 5 e j E ld Next Section 4 Workshop Module The Horizontal and Vertical Replication buttons together with the number in the Size box specify the direction and number of replicates to be automatically defined With the horizontal direction row button active and the Size set at 5 if you e Click a well the sample type is incremented by one for the next five wells in the horizontal direction
126. hen the Export to Excel command is selected from the menu the iQ5 software exports the selected data into a protected workbook that is automatically open by Microsoft Excel The protected workbook contains all of the formatting and fluorophore information separated into worksheets NOTE The numeric values contained in the protected workbook are exact values from the software application that include several non significant figures beyond the decimal point This is important to note when considering whether to transfer spreadsheets by a copy and paste command or the Export to Excel command With the copy and paste command only the significant digits displayed in the iQ5 software interface are transferred to Excel 6 9 4 Setting Analysis Parameters for Gene Expression Analysis Using the Settings Tab The Settings tab is used to assign gene and conditions parameters and efficiency values to be used in the calculations for gene expression analysis There are three sections of the Settings options window e Gene List e Condition List e Data Set List 6 9 4 1 Gene List Assigning Reference Genes and Target Specific Reaction Efficiency When the Settings button is active and Gene List is selected the genes being analyzed in the experiment are listed along with their full name This Full Name column is editable so that longer more descriptive names may be entered if your Gene Name entry was abbreviated NOTE Gene Name entries have a character
127. hese wells by typing your desired name into the gene pull down men then click enter to apply the name to the selected wells e Change the condition assignments in these wells by typing your desired name into the condition pull down menu then click enter to apply the name to the selected wells NOTE The Gene and Condition Names have a character entry limit of 15 characters Minimize the gene expression plate interface by clicking on the button to return to the standard view of the Gene Expr tab window Click the Settings tab then select Gene List to set your desired reference gene s Click Normalized expression ddCt Click Recalculate to see your results Normalized expression results are graphed The Data Table spreadsheet is accessed by clicking Data Table The Data Table spreadsheet lists the Condition and Gene name calculated expression values and C values Right click to print or export this data to Excel NOTE Use the Settings tab then click Gene List to enter a specific user defined gene reaction efficiency Use the Settings tab then click Condition List to select a particular sample as a control sample 6 9 2 Relative Quantity dC AC Relative quantity is the target sequence concentration relative to other samples in the experiment This is sequence quantity without taking into account reference genes for normalization No calculations are made to account for loading differences or differences in the number of
128. his paper Standard deviations may be slightly different How does normalized expression as calculated by this software compare to the model outlined by Dr Jo Vandesompele on the geNorm Web site The iQ5 software uses the models outline on the geNorm web site http medgen ugent be jvdesomp genorm and gives you the same results Why would I have to assign gene names in the Gene Expr tab If you have not assigned gene names in your initial plate setup or if you are studying more than 5 genes you can use the plate interface in the Gene Expr tab to assign these relationships Can I customize my gene and condition names Yes Both the gene and condition pull down menus will accept any text you type into them If your names are very long you can edit the name field in the gene and condition lists The long name will only be displayed in the legend above the graph The name is displayed on the x axis How do I determine reaction efficiencies Typically the efficiency for each primer or primer probe set is evaluated and recorded during assay development Generate a standard curve using serial dilutions of a representative sample across a relevant dynamic range and then record the efficiency for subsequent gene expression analysis 6 10 Gene Study Multi File Gene Expression Analysis When conducting gene expression experiments it may become necessary to run more than a single plate in order to analyze all required samples Similarly the
129. hore icon When the Delete Fluorophore icon is active and you click in a well in the plate setup the currently selected fluorophore is removed from all wells in that replicate group It does not change the definition of the sample type in that well Delete All icon When the Delete All icon is active and you click a well all information from that well is removed The other members of a replicate group are not affected 4 2 3 Editing or Creating a New Plate Setup 1 ME LEE EL o M Open the Plate Setup Editor Window within the Workshop module by using one of the following methods Click Create New in the plate setup display pane to enter the Plate Setup Editor or e Click Plate Select the desired plate setup file from the file tree directory Double click the file name to go directly to the Plate Setup Editor or e Click Plate Select the desired plate setup file from the file tree directory Click the file name to open the plate setup in the bottom right section of the Workshop window Click Edit to open the plate setup in the Plate Setup Editor or e Click Data File Select the desired data setup file from the file tree directory Click the file name to open the plate setup associated with the data file in the bottom right section of the Workshop window Click Edit to open the plate setup in the Plate Setup Editor Enter or edit any notes about the plate setup in the Notes box Enter or edit the sample volume seal type
130. iCycler thermal cycler with care and with clean dry hands during unpacking and assembly 1 2 1 System Checklist MyiQ2 Optics Module catalog 7170 9758 e Optics module e Power cord e iQ5 optical system software installation disk 170 9753 e Amplification tech notes CD version 1 0 iCycler Chassis catalog 170 8701 e Power cord e iCycler chassis e iCycler thermal cycler instruction manual Section 1 Getting Started iQ5 Optical Accessory Kit catalog 170 9752 e Optical reaction block e Modified sliding rear cover for iCycler thermal cycler e Serial cable e USB cable e Filter extraction tool e Optical tape applicator tools 3 e Optics support bracket e Support bracket screws e Hex driver e Hex screws e Spare part halogen lamp MyiQ2 Calibrator Dye Solution Kit catalog 170 8791 e 1x calibration dye solutions 4 dyes 3 tubes of each dye External Well Factor Solution catalog 170 8794 e External well factor solution 5 tubes Sample Consumables e PCR plates and sealers e Reagents Contact your local Bio Rad office if any system components are missing or damaged Accessories The following accessories are required to complete the installation e Scissors e 2 Phillips screwdriver e Calibrated micropipet s e Aerosol barrier pipet tips e Optical quality sealing film or tube caps e Optical quality PCR plates or tubes 1 2 2 Installing the Optical Reaction Module on the iCycler Chassis 1 Remove the existi
131. iQ Real Time PCR Detection System e Fluorescein FAM SYBR Green I Recommended fluorophores for the iQ5 Real Time PCR Detection System e Filter position 2 485 30X 530 30M Fluorescein FAM SYBR Green I e Filter position 3 530 30X 575 20M HEX TET VIC JOE 133 Section 9 Instrument Maintenance e Filter position 4 545 30X 585 20M TAMRA Cy3 e Filter position 5 575 30X 625 30M Texas Red ROX e Filter position 6 630 30X 685 30M Cy5 LC640 NOTE The filter designation 485 30X indicates that this filter will allow light between 475 and 595 nm to pass through The first number 485 indicates the center of the wavelength of light The second number 30 indicates the total breadth of wavelengths of light that can pass through it The letter X indicates that the filter is specified for excitation only and the letter M indicates emission only types of filters Excitation and emission filters are not interchangeable 9 2 2 Accessing the Filters To access the existing filters proceed as follows 1 Turn off the power to the optics module 2 Reach behind the optics module and unscrew the short fastener that secures the lid of the module in place 3 Using gentle pressure and both hands push inward on the rear vents located on the top half of the instrument casing Lift upward to remove the cover of the optics module 4 To access the excitation filter wheel remove the black plug from the slot located near the lamp at the right
132. ic preferences can be set for e File Paths e Plate Setup options e Protocol options e File Selection at application startup e PCR Quant module analysis settings 122 Section 8 User Profiles e Allelic Discrimination module analysis settings e End Point module analysis settings e Gene expression module analysis settings 8 1 1 File Paths Preferences Each file created by the iQ5 software is destined to a specific folder location on your computer this is known as a file path The File Paths preferences box Figure 8 1 can be used to select the folders that you wish to save your files into when logged into the iQ5 software The default File Paths for all users is a folder created with the current user s User Name File Paths Browse Protocol Paf y C Program Files Bio Rad iQS Users louise A Prog i lis i B utto n Plate Path C Program Files Bio Rad iO5 Users louise ea Run Set Path C Program Files Bio Rad iQS Users louise E Data File Path C Program FilesBio RadliQsYUserslouise Ex Gene Exp Pathic Program Files Bio Rad iQ5 Users louise Ex Fig 8 1 The File Paths dialog box File Paths can be specified for protocol files plate setup files run sets data files and Multi file Gene Expression Analysis Files Gene Exp Path To set the file path to an existing directory click the browse button and navigate to the appropriate folder To set the file path to a new directory click the browse button then
133. ied for the selected method and End Point Tolerance settings The following conditions result in an error message e If the Positives method is selected and there is not at least one positive control defined in the End Point analysis spreadsheet e If the Negatives method is selected and there is not at least one negative control defined in the end point analysis spreadsheet e If the Positives amp Negatives method is selected and there is not at least one positive control and at least one negative control defined in the End Point Analysis spreadsheet e If no Controls are defined e Ifthe range is less than one that is the negative control RFU average is larger than the positive control RFU average End point analysis cannot be performed if any of these error conditions is present when you click Recalculate Once you are satisfied with your end point analysis you may choose to view save or print a customized End Point Analysis report Click the Reports menu to obtain customized reports for the end point data Click Print to print the End Point analysis spreadsheet 6 9 Gene Expression Analysis The Gene Expression Gene Expr tab is used to evaluate relative differences in any target concentration For example you can evaluate relative numbers of viral genomes or stably transfected sequences The most common application is evaluating target concentration in cDNA samples to infer steady state messenger RNA levels 78 Section 6
134. ile name saved When collecting dynamic well factors the plate is held at 95 C for 2 5 minutes prior to the first cycle with a setpoint of 90 0 C or higher 5 2 2 Persistent Well Factors When setting up a plate setup to run the experiment specific reaction vessel and sealing methods are chosen This plate setup will not be able to be used to run an experiment unless persistent well factor and background calibration files with matching vessel and sealing type have already been collected 42 Section 5 Run Time Central Module Persistent well factors are stored in the Well Factors folder in the iQ5 Folder Refer to section 7 for information on the calibration and generation of persistent well factors In general persistent well factors can be used for approximately three months but should be re generated anytime something pertinent to the optical system is changed such as the optical filters or the camera lamp A warning reminder to re calibrate to generate new persistent well factors will occur when Persistent Well Factors are older than 90 days 5 3 Real Time PCR Experiments Using DNA Binding Dyes In most real time PCR experiments using DNA binding dyes like SYBR Green I or ethidium bromide dynamic well factors may not be collected When the template DNA is denatured the fluorescence of these dyes is not sufficiently high to calculate statistically valid well factors using the experimental plate There are three possible solu
135. ils about the data displayed in the PCR Quant screen These optional details will be displayed in the results spreadsheet of the PCR Quant screen and include the following information about each sample Identifier Concentration Threshold Cycle and End Point Call 126 Section 8 User Profiles 8 1 6 Allelic Discrimination Module Preferences The Allelic Discrimination preference box Figure 8 7 can be used to set the default display conditions used in the Allelic Discrimination screen The Display Mode can be set to Threshold Cycle or RFU using the radio buttons The radio buttons for the Normalize Data option become active only when the Display Mode is set to RFU Allelic Discrimination Display Mode Threshold Cyce RFU Normalize Data ves no Fig 8 7 Defining Allelic Discrimination Display Options 8 1 7 End Point Module Preferences The End Point module preference box Figure 8 8 allows you to predetermine several analysis and display options associated with analyzing final RFU in an end point analysis assay End Point Method Negatives v Ranks 10 End Cycles to Avg PcR s End Point Only Run2 Tolerance Mode RFUs Tolerance 96 10 0 Color Scheme Yellow Green hd Fig 8 8 Defining Analysis and Display Options for the End Point Module e Method The Method box allows you to select the method of assigning positive and negative values to your unknowns based on RFU values The Method box consis
136. in the biological system being studied Normalized expression is the relative quantity of your gene normalized to the relative quantities of the reference gene s Simply stated the quantities of the reference genes are used to normalize the values of your genes of interest Provided the reference genes are not regulated in your system normalized expression calculations will account for loading differences or variations in cell number represented in each of your samples This value is sometimes referred to as ddC or AAC because of the equation initially introduced by Livak et al 1995 to evaluate normalized expression The software uses a modification of this equation Normalized expression can be viewed as normalized target sequence quantity if your assay has included controls for this purpose To calculate Normalized Expression ddC 1 In the PCR Quant screen with a opd data file open assess Threshold and Baseline information for the data file and make changes if necessary 2 Click the Gene Expr tab 80 3 hg Ur R Section 6 Data Analysis Module Make any changes to Gene and Condition for example Sample and Treatment assignments in the gene expression plate interface e Expand the gene expression plate interface view by clicking on the button to make well identification and selection easier Highlight the wells in the gene expression plate interface you wish to edit e Change the gene assignments in t
137. in user preferences only fluorophores that are compatible with the connected instrument will be displayed If only fluorophores that are not available for detection by the connected instrument are selected by default the Plate Setup window will display FAM only 8 1 3 Protocol Preferences The Protocol preferences box can be used to set the defaults for the current user for Melt Curve Gradient and Well Factor Collection Type Figure 8 4 Protocol Melt Curve Set Point 55 0 EZ End Temp 5 0 ET Temp Change p 5 ES Gradient Set Point 55 0 Ranger 10 0 WellFactor Collection Type Persistert Q Dynamic Fig 8 4 Defining User Specific Protocol Preferences The following default conditions can be set for the Protocol e Melt Curve Enter the desired beginning temperature Set Point ending temperature End Temp and the change in temperature that will occur at each repeat Temp Change Click Save e Gradient Enter the desired lowest temperature Set Point and the Range for the gradient in the appropriate text boxes or use the scroll buttons to enter these values Click Save Well Faction Collection Type Select the radio button associated with the well factor that you wish to use as the default type Click Save If Dynamic is chosen as the default type dynamic well factors will be used unless restricted by the Plate Setup 8 1 4 File Selection at Application Startup Preferences The file that you wish to have auto
138. including e 2Step tmo This protocol can be used for most single fluorophore or SYBR Green I real time PCR experiments e 2StepAmp Melt tmo This protocol can be used for most SYBR Green I real time PCR experiments The melt curve that follows the qPCR experiment can be used to determine the number of amplicons produced in the qPCR reaction e Gradient tmo This protocol can be used to determine the most optimal temperature to perform real time PCR experiments 4 4 Run Set A Run Set is a linked pairing of a Plate Setup and Protocol files Run Set files generated by the iQ5 software are stored with the extension run Run sets are useful for experiments that are repeated on a frequent basis When the Run Set tab is selected run set files are shown in the file browser The protocol defined in the run set is shown in the Selected Protocol pane and the plate setup defined in the run set is shown in the Selected Plate Setup pane Figure 4 17 237 Section 4 Workshop Module EN Setup WEN Plate Summary EEN Protocol MEN Plate EZ Runset MMM Data File selected Run Set Hex_FAMRunSetsun Masks ar Hex FAMRunSet run NE Persisten Sample files 41 RepottTe SS 8 3 RMEDate SampleFil Run End Point Supporti Users admir DP WalEant M Analyze lt gt Selected Protocol 2Step tmo within Hex_FAMRunSetrun Selected Plate Setup Hex FAM pts within Hex FAMRunSetrun
139. ing the printer icon opens the Windows Print dialog box Click OK to complete the printing task The Export to Excel command on the spreadsheet menu is useful for exporting exact values from the spreadsheet When the Export to Excel command is selected from the menu an Export to Excel file save box is displayed Choose a location of where the Excel file is to be saved and click Save The iQ5 software automatically exports the selected data into a protected workbook The protected workbook generated by the iQ5 software contains the text values of what is represented on the spreadsheet For example checkboxes from the software application are replaced by True or False text in Excel The numeric values contained in the protected workbook are exact values from the software application that include several non significant figures beyond the decimal point This is important to note when considering whether to transfer data by a copy and paste command or the Export to Excel command With the copy and paste command only the significant digits displayed in the iQ5 software interface are transferred to Excel 91 Section 6 Data Analysis Module 6 9 7 Graphing Options for Expression Data Graph Expression Relative to Control or Relative to Zero These options shown in Figure 6 50 allow you to present data with bars originating at 1 relative to control or at zero relative to zero If you assign a control in your dataset selecting the
140. ion system for the first time mask alignment background calibration persistent well factor collection and pure dye calibration for MyiQ2 and iQ5 systems only must be performed See section 7 Calibrating the Instrument for detailed instructions on calibration 1 6 Compatibility with Earlier Versions of the iQ5 Optical System Software Data Files Protocol plate setup and data files generated or saved with version 3 0 or 3 1 of the iQ Real Time Detection System software and earlier versions of the iQ5 software are recognized by version 2 1 of the iQ5 software These tmo pts and opd files can be opened from within iQ5 software for analysis To open or import files generated by older versions of the iQ Real Tme Detection System software version 2 3b and earlier you must open the file in version 3 0 or 3 1 of the iQ software first and then save the file again Calibration Files Earlier versions of the iQ5 Optical System software earlier than version 2 1 have volume dependent calibration files for both persistent well factors and pure dye calibration collection In version 2 1 there is no volume dependency for these calibration files If a computer is upgraded from an earlier version of the iQ5 Optical System software to version 2 1 existing calibration files will be upgraded to remove calibration volume dependency For example Persistent_Plates_Film_25ul_lQ2Emulator_IQ2Emulator xml will be upgraded and renamed as Persistent
141. is as the time appears in the spreadsheet Alternatively 10 seconds can be entered as 0 10 5 Click in the PCR Melt Data Acquisition column and select Melt Curve from the pull down menu A green camera will appear in the Data Acquisition cell and two additional columns entitled Temperature Change and End Temperature will be added to the spreadsheet By default the temperature change is set at 0 5 C and the end temperature is at 95 C unless the setpoint is 95 C The number of repeats to achieve this melt curve is automatically calculated The temperature change can be as low as 0 1 C increments Typical temperature change values are 0 3 0 59C for SYBR Green I 36 Section 4 Workshop Module Cyde Repeats Step pn Sathish ie eee LUE 1 1 1 1 00 95 0 2 1 1 1 00 55 0 3 81 1 Ui sso aw MekCuve v 05 i Fig 4 16 Melt Curve Protocol Editing Table 6 Anneal curves can be also created by entering the temperature change as a negative value for example 0 5 7 Save the protocol by entering a file name and then clicking Save amp Exit Protocol Editing NOTE The minimum dwell times necessary for data collection are 10 seconds for 1 fluorophore We recommend using a slightly higher dwell time than the minimum values so that more data points are collected at each repeat 4 3 6 Sample Protocol Files There are several sample protocol files that can be used as editable templates for custom protocol design
142. is module 2 At the top of the Data Analysis window click Edit Plate GEB Est Plate 3 A modified version of the Plate Setup Editor will open Figure 6 63 In this modified window you cannot add fluorophores to or remove fluorophores from the fluorophore list Nor can you add a previously defined fluorophore or remove a previously defined fluorophore from a well sii r Editing Plate Sample Volume ES Apply Plate Changes s Seal Ty H m ms zii Cancel s Vessel Type 5 Spreadsheet zm e NI ES DN Experiment Name qm WAR NA My experiment zi IReplicates 1 2 3 4 5 6 7 8 9 10 11 12 Fluorophore Probe Primer FAM E BOOSIE 123 Dj HEX B 5 WM s Miis 1 1 jz c Mis 10 e 1 5 D 2 6 M10 1 Size z mji 3 7 11 1 1 I whole Plate loading Next F 3 7 1 a 1 Units copy number xj Applyunitsto Whole Plate Fluorophore G 4 Wis W 1 1 H 4 ais H2 1 t E Scientific Notatin W Dilution Series Row Column SampleType Rep Identifier Condition Quantity Units A 2 v 1 NA copy number Unknown DA 1 NIA copy number Fig 6 63 The Edit Plate Window 4 Edit any notes about the plate setup in the Notes box Edit the name of the experiment in the Experiment Name box 6 For most experiments the Whole Plate Loading box will be checked With Whole Plate Loading changes ma
143. it of 15 characters e Minimize the Gene Expression Plate interface by clicking on the button to return to the standard view of the Gene Expr tab window Click Relative Quantity dCt Click Recalculate to see your results Relative Quantity results are graphed The Data Table spreadsheet is accessed by clicking Data Table The Data Table spreadsheet lists the Condition and Gene name calculated expression values and C values Right click to print or export this data to Excel NOTE Click the Settings tab then select Gene List to enter a specific user defined gene reaction efficiency NOTE Click the Settings tab then select Condition List to select a particular sample as a control sample If you need to compare these data to results obtained in other opd files you will need to enable this file for Multi file Gene Expression analysis also called a Gene Study To enable your file for Gene Study 1 2 Click the Enable for Gene Study button Go to the File menu to save your file Calculating Normalized Expression ddC AAC To calculate Normalized Expression ddC 1 In the PCR Quant screen with a opd data file open assess Threshold and Baseline information for the data file and make changes if necessary Click the Gene Expr tab Make any changes to Gene and Condition for example Sample and Treatment assignments in the Gene Expression Plate interface e Expand the Gene Expression Plate Interface view
144. ith your unit However the optical filters are removable and user serviceable by design Handle the optical filters with care as they can crack if dropped Also avoid touching the surfaces of the filters especially with fingers as this can impair data quality Contact Bio Rad for replacement filters 134 Section 9 Instrument Maintenance You may clean the optical filters by wiping them gently with lens paper and 70 ethanol You may obtain lens paper and lens cleaning solution by ordering a Lens Cleaning Kit from Bio Rad catalog 170 7731 After cleaning both sides of an optical filter hold it up to the light and make sure that no smudges fingerprints debris or water marks remain Once cleaned reinsert the optical filters as described above 9 3 Replacing the Lamp When replacing the lamp you must only use lamps supplied by Bio Rad Bio Rad lamps are subject to additional tests and standards geared specifically toward real time data collection Lamps from alternative sources which may appear to be similar may not deliver the same optical quality performance and lifetime as those supplied by Bio Rad If the camera is to remain continuously on we recommend the lamp be replaced every 6 months NOTE When a lamp is overdue for replacement if may flicker sporadically causing a wavering of the data in all wells simultaneously Installing a new lamp will alleviate this problem Caution Take care when changing the lamp as it m
145. ither PCR Base Line Subtracted or PCR Base Line Subtracted Curve Fit You may alter the analysis mode of the source data in the PCR Quant tab within the Data Analysis module Current method Displays the currently selected method for assigning positive and negative values to unknowns Tolerance Displays the currently selected mode of tolerance calculation Immediately beneath the displayed mode is the tolerance value If RFUs are chosen then the Set Tolerance in RFUs is given If Percentage of Range is chosen then the Calculated Tolerance in RFUs appears Range The value displayed here is dependent on the method chosen e If the Positives method is chosen the Range is the Positive Controls Average RFU minus the lowest RFU e If the Negatives method is chosen the Range is the highest RFU minus the Negative Controls average RFU e If the Positives amp Negatives method is chosen the Range is the Positive Controls average RFU minus the Negative Controls average RFU Controls Average RFUs Displays the average RFU of all positive controls No value is shown if there are no positive controls defined Control Tolerance In the Positives or Positives amp Negatives methods samples with values equal to or higher than this amount will be called as Positive No value is shown if there are no positive controls defined Controls Average RFUs Displays the average RFU of all negative controls No value is shown if there are no negat
146. ive controls defined Control Tolerance In the Negatives method samples with values equal to or higher than this amount will be called as Positive In the Positives amp Negatives method samples with values less than this amount will be called Negatives No value is shown if there are no negative controls defined 6 8 2 End Point Analysis Spreadsheet The End Point analysis spreadsheet contains the necessary data to perform end point analysis and also displays the values assigned to unknowns after Recalculate is clicked The table headings include the following Well Lists the well ID of each sample You may click on the Well column header to sort the table by wells Note that you may also include or exclude certain wells from end point analysis via the Analyze Wells button above the spreadsheet Sample Type Lists the sample type of every well as defined in the plate setup You may click on the Sample Type column header to sort the table by sample type End RFUS Lists the absolute RFU averages for each well as calculated from the end cycles which are specified in the End Cycles to Average field You may click on the End RFUs column header to sort the table by end RFUs 77 Section 6 Data Analysis Module e Define Controls Lists any positive or negative controls defined in the original plate setup file You may also add new controls or edit existing controls in this column by clicking on the drop down menu on the right sid
147. kshop Navigate the directory until the desired data file is found Double click the file name to bring the file directly into the Data Analysis module or e Click Data File above the directory of the home workshop Navigate the directory until the desired data file is found Click the file name once to open the plate setup associated with the data file in the bottom right section of the Workshop window Click Analyze to bring the data to the Data Analysis module 2 At the top of the Data Analysis window click Edit Plate 3 A modified version of the Plate Setup Editor will open In this modified window you cannot add fluorophores to or remove fluorophores from the fluorophore list Nor may you add a previously defined fluorophore or remove a previously defined fluorophore from a well 4 Edit any notes about the plate setup in the Notes box Edit the name of the experiment in the Experiment Name box 6 For most experiments the Whole Plate Loading box will be checked With Whole Plate Loading changes made to any fluorophore within a well are extended to the other fluorophores within the well and within the replicate group If you are editing a plate the Whole Plate Loading checkbox may be unavailable because it is not appropriate based on the current definition of the plate 7 Click a fluorophore 16 10 11 12 13 Section 2 Quick Guides Click a sample type icon Select the type of replicate loading desired
148. l plate fails 7 2 Components Required for Calibration The following reagents and accessories are required to calibrate the MyiQ2 instrument e MyiQ2 Calibrator Dye Solution kit catalog 170 8791 e External Well Factor Solution catalog 170 8794 e 3 x 96 well PCR plates or preferred reaction vessel e Optical quality sealing tape or preferred sealing method 111 Section 7 Calibrating the Instrument The following reagents and accessories are required to calibrate the iQ5 instrument e iCycler iQ calibrator dye solution kit catalog 170 8792 which contains iQ5 specific pure dye solutions and external well factor solution e 3 x 96 well PCR plates or preferred reaction vessel e Optical quality sealing tape or preferred sealing method NOTE Calibrator dye solutions are NOT required for calibrating the MyiQ system 7 3 Preparing Calibration Plates Preparation of an External Well Factor Plate Preparation of this plate is required for mask alignment and persistent well factor generation for the MyiQ2 MyiQ and iQ5 systems To prepare an external well factor plate 1 Dilute the 10x External Well Factor Solution to 1x with water 2 Pipet 25 ul of the 1x external well factor solution into each of 96 wells of your preferred reaction vessel 3 Seal using your preferred sealing method you will use in your future experiments Preparation of a Background Calibration Plate Preparation of this plate is required for background ca
149. lar Probes Inc to sell reagents containing SYBR Green I for use in real time PCR for research purposes only Cy is a trademark of GE Healthcare SYBR and Texas Red are trademarks of Molecular Probes Inc HEX TAMRA TET and VIC are trademarks of Applera Corp Excel Windows and Windows XP are trademarks of Microsoft Corporation Phillips is a trademark of Phillips Screw Company Corporation Safety Information Grounding Always connect the MyiQ 2 MyiQ or iQ 5 optics module to a three prong grounded AC outlet using the AC power cord provided with the system Do not use an adaptor to a two terminal outlet Always ensure that you set the module power switch to the off position when you connect or disconnect power cords Handling Handle all components of the real time PCR detection system with care and with clean dry hands at all times The optical system contains mirrors and lenses that may shatter if the unit is dropped or struck with great force If the unit is damaged such that internal components or wires are exposed contact your local Bio Rad office immediately Do not attempt to repair or power on the instrument Servicing The only user serviceable parts of the optics module are the lamp and filters Call your local Bio Rad office for all other optics module and thermal cycler related service When you replace the lamp or filters open only the outer casing of the optics module The camera lamp may get extremely hot
150. lculated as follows Cr MIN T Cr sample Relative Quantity ampie cene Ecenex Where E Efficiency of primer primer probe set this efficiency is calculated as follows Efficiency 0 01 1 where 100 2 Cr min Average Cr for the sample with the lowest average Cr for gene x Cr sample Average Cr for the sample 97 Section 6 Data Analysis Module Relative Quantity When a Control is Assigned With a control assigned relative quantity dC for any sample for all genes is calculated as follows Cr contrat Cr sample Relative Quantity ampie Gene x Genes Where E Efficiency of primer primer probe set this efficiency is calculated as follows Efficiency 0 01 1 where 100 2 Cr control Average Cr for the sample which has been assigned as a control Cr sample Average C for the sample This is where the calculations differ from those outlined by Dr Jo Vandesompele on the geNorm web site http medgen ugent be jvdesomp genorm In the example on the geNorm Web site the results are not scaled the control until normalized expression is calculated This is referred to as rescaled normalized expression in the example spreadsheet Standard Deviation of Relative Quantity of Gene x for a Given Sample SD Relative Quantity SD C sample Relative Quantity sample Ln E Gene x Where SD Relative Quantity Standard Deviation of the Relative Quantity SD C sample Standard Deviation o
151. le is being analyzed When you click on the Edit Study button the iQ5 software will display the Gene Expression Study Manager Individual files can be added to the Gene Study by clicking Add opds To remove files from the current Gene Study To select or deselect an individual file click the checkbox within the 102 Section 6 Data Analysis Module corresponding row If one or more files have been selected clicking the Remove button will remove those file s from the Gene Study The Notes field is also editable from the Gene Expression Study Manager Once the desired changes have been made click OK to close the Gene Expression Study Manager and return to analysis of the Gene Study file in the gene expression module Remember to immediately save your edits to the Gene Study before continuing with your analysis NOTE The Edit Plate module of the iQ5 software is not available when a Gene Study file extension gxd is opened All plate information changes to the data in a Gene Study that is sample ID must be made from the plate interface of the Gene Expression window 6 10 5 Excluding Including Samples Using the Gene Expression Plate Interface When a Gene Study file is displayed the Analyze Wells button that is available when viewing a single Gene Expression file is no longer displayed In its place is the Include Sample Exclude Sample drop down list located above the plate interface Figure 6 60 Include Sample Y Include
152. lelic Call Q allele 1 control 1 allele 2 control 2 deterozyote unknown None Fig 6 32 The Manual Call Specification Options Box 2 Select one of the allelic call types by clicking its radio button 3 On the plot drag the cursor around the well or wells to be assigned the call When the mouse button is released the new assignment will be made on both the plot and the data spreadsheet To make manual calls in the data spreadsheet 1 Click Manual Call 2 Click the cell for the desired well in the Call column of the data spreadsheet Select the new call from the pull down menu 3 Note that unknown may only be selected in Manual Call mode 6 7 6 Display Mode Choose the aspect of the data that will be analyzed by clicking either Threshold Cycle or RFU Figure 6 33 Display Mode Threshold cycle rru Fig 6 33 The Display Mode Option Buttons When you click RFU the Select Cycle option Figure 6 34 appears so you can analyze RFU data from any cycle Change the cycle by entering a new value in the Select Cycle drop down list ey ice Section 6 Data Analysis Module Select Cycle Fig 6 34 The Select Cycle Drop Down List Box 6 7 7 Vertical Threshold The Vertical Threshold box Figure 6 35 displays the current position of the vertical bar This box is not editable The positions of the vertical bar along with the position of the horizontal bar determine the genotype assignment fo
153. les you will need to enable this file for Multi file Gene Expression analysis also called a Gene Study To enable your file for Gene Study 1 Click the Enable for Gene Study button 2 Goto the File menu to save your file Gene Study Multiple File Gene Expression Quick Guide To create a Gene Study for analysis of multiple opd files 1 For each opd file you wish to combine to a Gene Study a In the PCR Quant screen with a opd data file open assess Threshold and Baseline information for the data file and make changes if necessary b Click the Gene Expr tab c Make sure all files to be included in the Gene Study have the Enable for Gene Study button active d Save each individual file 2 From the menu toolbar select File New Gene Study 3 Inthe Gene Expression Study Manager select Add opd s 4 Select the files that you wish to add to the Gene Study More than one file can be selected 5 Once the files are added to the Gene Expression Study Manger select OK 6 Make any changes to Gene and Condition for example Sample and Treatment assignments in the Gene Expression Plate interface e Expand the Gene Expression Plate interface view by clicking on the button to make well identification and selection easier Highlight the wells in the gene expression plate interface you wish to edit e Change the gene assignments in these wells by typing your desired name into the gene pull down men then click ent
154. librations for the MyiQ2 MyiQ and iQ5 systems Leaving the wells completely empty seal 96 wells of your preferred reaction vessel with your preferred sealing method Preparation of the Pure Dye Calibration Plate Preparation of this plate is required for pure dye calibration for the MyiQ2 and iQ5 systems To prepare a pure dye calibration plate 1 In clean wells pipet 25 ul of the appropriate 1x calibrator pure dye solution into 8 wells of your preferred reaction vessel 2 Repeat for all fluorophores you will use in your future experiments See example of Pure Dye Plate Setup 3 Seal using your preferred sealing method you will use in your future experiments NOTE Pure Dye Calibration is NOT performed on the MyiQ Real Time PCR Detection System 7 4 Performing Calibrations The MyiQ2 MyiQ and iQ5 systems are calibrated using the calibration plates described in section 7 3 above and using the Calibration Module of the iQ5 software Allow the instrument optics to warm up for 20 min prior to performing the mask alignment background calibration persistent well factor collection or pure dye calibration procedures 112 Section 7 Calibrating the Instrument Calibrations are performed in the order as they appear in the tabs of the Calibration module e Mask Alignment e Background Calibration e Persistent Well Factor Generation e Pure Dye Calibration 7 4 1 Performing Mask Alignment Before you align the mask complete t
155. lines and threshold will be conducted including every defined well If the file was previously saved after an analysis the last set of analysis conditions are applied again Make any manual adjustments to the threshold by clicking and dragging the green threshold line Make any manual adjustments to the baseline by right clicking on the amplification traces plot to access the baseline threshold parameters popup window and editing User Defined options NOTE You can revert to software auto calculated threshold and baseline by right clicking on the amplification traces plot to access the baseline threshold parameters popup window and selecting Auto Calculated 2 4 2 End Point Quick Guide You can implement End Point analysis in two ways Click Run End Point to initiate the collection of End Point data from a sample plate Click the End Point tab in the Data Analysis module for an existing data file Initiating an End Point Run 1 2 Insert the experimental plate into the iCycler reaction module From the Workshop Setup Window Select or Create the Plate Setup from the Plate Setup tab 3 Click Run End Point Check that the desired Protocol and Plate Setup are displayed in the bottom half of the Initiate Run screen In the Run Time Central Initiate Run tab specify the setpoint for data collection and then click Begin Run NOTE You must use Persistent Well Factors for every End Point Run Name the file with a unique name in
156. located at the rear of the iCycler thermal cycler Connect the serial cable to the rear of the iCycler chassis and to the serial port on the side of the optics module This connection enables communication between the iQ5 software and the iCycler thermal cycler e USB port connector Connect the supplied USB cable between the optics module and a USB 2 0 high speed enabled port on the computer Data are transferred to the computer via this cable This single connection directs the operation of both the optics module and the iCycler thermal cycler by the iQ5 Optical System software e Atthe right rear corner of the optical reaction block is a single connector the Positive docking connector This self aligning connector is secured into place when the optics 1 3 Section 1 Getting Started module is installed on the iCycler chassis This connection senses when the lid handle is lifted Installing the iQ5 Software Locate the software installation disk for iQ5 Optical System software version 2 1 provided with the MyiQ2 system This installation disk is compatible with computers running the Windows XP and Windows Vista 32 bit operating system 1 2 Insert the iQ5 Optical System software installation CD in a CD ROM drive If the installation program does not begin automatically click Run in the Start menu and then type X iQ5 Setup where X is the drive letter of the CD ROM drive For example if the CD ROM is the E drive type E iQ5
157. m AllReplictes RandomColors Remove Symbols le Leal Led IRSE Le ICO ICICI Fig 6 20 The Define Trace Style Window To modify a trace style 1 Select the Sample Types Group to be modified for example Standards Note that green is the default color that is automatically assigned to the Standards group when this button is active 2 To specify a different color for a given sample type group on the plate click on the colored button in the Select Color column Select the symbol type the default style is None 4 Click Preview to see your changes and if those changes are satisfactory click OK To choose on a well by well basis click the Selected Well s button select a color and symbol type and then select the desired wells in the plate grid at the bottom of the Define Trace Style dialog box Click a column or row header to change an entire row or column The dialog box outlines the edge of each well in the grid in the selected trace color Select All Replicates to apply the selected color to all selected replicates of the wells To reset original trace style settings click Random Colors and Remove Symbols then click OK Display Data Option Click Display Data to change the view of the results spreadsheet as follows e When Display Data is unselected the results spreadsheet displays the plate spreadsheet view e When Display
158. m 14 different units of measure to match the units used in your experiment Seal Type Chose from Film Domed Cap and Flat Cap Vessel Chose from Plates Strips tube strips and Tubes Sample Volume Set the sample volume in ul that will be used as the default value Dilution Series Set the defaults for the dilution factor and specify whether the series is increasing or decreasing in quantity You may specify if scientific notation is to be used when the dilution series values are displayed Replicate Size Used to specify the default replicate size Replicate Loading Used to specify the default direction replicates will be loaded You can choose from Row replicates added horizontally Column replicates added vertically and Row and Column replicates added in a block See Specifying Replicates for more information Calibration Plate This is used to set the default file to be selected for the pure dye calibration plate This file will be used after Application Startup Fluorophores Place a checkmark next to the fluorophores that you wish to have automatically selected when creating a new plate setup No more than 5 fluorophores can be selected At least one fluorophore must always be selected 124 Section 8 User Profiles IMPORTANT NOTE The Fluorophores preference box will display all fluorophores available to the MyiQ2 MyiQ and iQ5 systems If both dyes that can and cannot be detected by the connected instrument are selected
159. matically selected when the iQ5 software is opened is determined using the File Selection at Application Startup radio buttons Figure 8 5 File Selections at Application Startup Last File Used Data File kRun Set Protocol Plate Data File D LSefton12 05 Denali Sample Files For v2 01Sample Alleli Fig 8 5 Selecting Custom File Defaults for Application Startup 125 Section 8 User Profiles e Last File Used This option will select the last Protocol Plate Setup Run Set or Data File used as the file to display in the Workshop screen when the iQ5 software starts up e Data File If you wish to have a specific data file selected when the iQ5 software opens select Data File and then use the browse button to navigate the data file that you wish to have selected on application startup e Run Set If you wish to have a specific Run Set file selected when the iQ5 software opens select Run Set and then use the browse button to navigate the run set file that you wish to have selected on application startup e Protocol Plate If you wish to have a specific protocol or plate setup file selected when the iQ5 software opens select Protocol Plate and then use the browse button to navigate either the protocol or plate file that you wish to have selected on application startup 8 1 5 PCR Quant Screen Preferences The PCR Quant preference box Figure 8 6 can be used to set the defaults for the PCR Quant screen You can set the default
160. med by activating the Enable for Gene Study button found in the Gene Expression window of the Data Analysis module Figure 6 57 Gene Name Y Condition Name v um Copy condition to all data sets EA Enablefor Gene Sudy Analyze Wells Fig 6 57 The Enable for Gene Study button is located above the plate interface section of the Gene Expr tab window When a file has been saved with the Enable for Gene Study button active it allows the saved file to be added to a Gene Study The default setting is that this button is not enabled You must save your data file after activating this option from within the Gene Expr tab window Gene Study Errors The file must be saved with the Enable for Gene Study button selected If a file with the Enable for Gene Study is not selected and there is an attempt to add the file to a Gene Study an error will be received Figure 6 58 Bio Rad iQ5 Selected data file SYBR_Timecoursel opd is not enabled for Gene Study To enable the datafile for Gene Study select the Enable for Gene Sudy button on the Gene Expr screen and save changes to the opdfile Fig 6 58 Gene study error message indicating that the file was not enabled To correct this error 1 Click OK to clear the error message 2 Re open the file that will be added to the Gene Study 3 Confirm that all wells to be analyzed have valid Cr values and activate the Enable for Gene Study button 4 Savethe file Attempt to add that
161. mmary The Plate Summary window shows well definition one fluorophore at a time and provides a more detailed view of the plate including standard quantities and identifiers as shown in Figure 4 12 It is useful to check the Plate Setup after editing or creating the Plate and also before performing a run It can also be used to check the plate layout in a data file The Plate Summary window can be accessed by e Clicking the Plate Summary tab from the Plate Editor window e Selecting a Plate Setup Run Set or Data File and clicking the Plate Summary tab from the Workshop module main window If the Data File tab is selected the plate shown in the Plate Summary window depends on whether Original or Current is clicked in the Selected Plate Setup window Original is the plate setup that the data file was created with Current is the last saved plate setup Setup Plate Summary aS 1 2 3 zi 5 6 rj 8 9 10 SampleType A copy number Identifier SampleType B copy number Identifier SampleType o copy number Identifier SampleType D Concentration Identifier SampleType m Concentration Identifier SampleType T7 copy number Identifier SampleType G copy number Identifier SampleType H copy number Identifier Fig 4 12 The Plate Summary Tab The spreadsheet displays data one fluorophore at a time Use the fluorophore buttons to
162. monitor that fluorophore and perform a pure dye calibration for that fluorophore E Section 4 Workshop Module 4 2 5 Whole Plate Loading There are two pieces of information required for each well sample type and fluorophore s to be monitored There are two ways of providing that information to the plate setup editor When Whole Plate Loading is selected you specify the sample type and fluorophores separately EA Whole plate loading e In Whole Plate Loading changes made to any fluorophore within a well are extended to the other fluorophores within the well upon pressing Enter or clicking in another cell of the spreadsheet These changes are then extended to every other well within the replicate group This is the most efficient way of defining a plate setup for a multicolor experiment when all wells have the same sample type at each dye layer monitored fluorophore For example you might choose to monitor four fluorophores in well A1 and in each dye layer the sample type is Unknown Note that if you choose Whole Plate Loading it is assumed that standards in each dye layer are at the same concentration that is the FAM standard might be at 500 copies and so by definition the HEX standard is also at 500 copies e When the sample types vary within a well or in the special case of standards when the concentration of standard varies with the dye layer deselect Whole Plate Loading When Whole Plate Loading is deselected you specify t
163. mplification Data RFU tab at the bottom of the spreadsheet view see Figure 6 11 The spreadsheet can display data for only one fluorophore The well number appears at the top of the spreadsheet and the cycle number appears down the side of the spreadsheet In Single Point mode each cycle is represented by one point that is the mean of all the data points analyzed during that cycle In All Candidates mode each individual data point displays 6 5 Amplification Plot Context Menu You can access PCR amplification plot data analysis and display parameters by right clicking on the PCR amplification plot After you right click on the plot the amplification plot menu displays Figure 6 12 Set Data Analysis Window Digital Filter BaseLine Threshold Single Point 4 Adjust Graph Define Trace Style Display Data Restore Graph Show All Traces Copy Graph Print Graph Print Amplification Data Print Std Curve Data Fig 6 12 The Amplification Plot Menu 6 5 1 Data Analysis Options There are three additional data analysis options in the amplification plot menu e Set Data Analysis Window e Digital Filter e Baseline Threshold Set Data Analysis Window After you click Set Data Analysis Window in the Amplification plot menu the Set Data Analysis Window appears as shown in Figure 6 13 The number of data points collected per cycle depends on the exposure and the dwell time of the cycle You can change the percentage
164. n End Point run by selecting a Plate Setup then click Run End Point The software transfers to the Initiate Run window in the Run Time Central module An End Point run uses a predefined canned protocol in which only the temperature of the data collection step can be modified Enter the desired temperature of the data collection step into the Setpoint box as shown in Figure 5 3 Click Enter Changes entered into the Setpoint box appear in the Selected Protocol region of the Initiate Run window 43 Section 5 Run Time Central Module Notes MS End Point Protocol and user selected ate Setpoint ko C Fig 5 3 The End Point Setpoint Box To begin a run click Begin Run The Save dialog box opens Type a name for the optical data file The iQ5 software saves data automatically during the experiment 5 5 Show Plate Window The Show Plate window Figure 5 4 can be used to visualize samples loaded into the reaction block of the system Use the Show Plate window to verify the position and orientation of samples prior to starting a run in the MyiQ2 MyiQ or iQ5 instruments Initiate Run E Show Plate Monitor Run Loaded wells validation for current plate Sample Gene Expression pts AM SUITS go 2048 Expose E Show loaded wells Well Average Inner Fluorescence Instructions D Click the Expose button nods a exposure An image of the sample area juorescence will b
165. n run a dialog box appears with the message Pure Dye Calibration Run Complete 4 Click OK to exit NOTE if you wish to calibrate for more than one vessel and sealing type combination repeat the process above to collect additional dye calibration files During a run the software will automatically use the correct file for the vessel and sealing parameters specified in plate setup NOTE Pure dye calibration is not required for SYBR Green dye designators SYBR and SYBR1 SYBR5 This completes calibration of the camera on the MyiQ2 and iQ5 systems Mask Background Well Factors a Pure Dye Pure Dye Plate Setup ron Se Masks A BioRadDefautNewPlatetestpts ar Tet Persistence Referto Help File for instructions on howto run this type of calibration H E Report Templates RMEData Samplefiles C3 SupportFiles ir Copy of TemplateFAM HEXpts it Ter ae Hex FAM pts MyiQ2 Puredye calibration pts se Fer 2 SYBR_Timecourse 1 pts s emn flue SYER Tmecouse2 pts Rin Pure Dye Calibration lt jm j i gt Selected Plate Setup MyiQ2 Puredye calibrations Edit Create New Sample Volume 25ul Seal Type Film Vessel Type Plates or Jee Jow jen i T LS A Lo v ez V ra AE d et S0 To iis A Vr d z e mo o ovo Fig 7 5 The Pure Dye Calibration Window 117 Section 7 Calibrating the Inst
166. nd then click Delete Selected Peaks You can press the Shift key to highlight multiple peaks and delete them all at once Delete Selected Peaks 6 6 3 Edit Melt Peak Begin End Temperature You may edit the begin and end temperatures for a melt peak by clicking Edit melt peak begin end temps as shown below E Edit melt peak begin end temps After you click this option button the currently active peak displays alone with a Begin Temp bar and an End Temp bar Drag the bars to the desired begin and end temperatures As you drag the begin and end bars along the Melt Peak plot the iQ5 software tracks the movement on the melt curve chart and updates the movement in the spreadsheet When the Edit melt peak begin end temps option is activated the Display Wells and Analyze Wells dialog boxes are unavailable In Figure 6 25 the peak begin temperature is 59 C the peak end temperature is 79 C and the threshold is 1 67 Section 6 Data Analysis Module Melt Peak Chart LL Fig 6 25 A Melt Peak Chart 6 6 4 Melt Curve Peak Control Area The control area appears between the spreadsheet and the plots This area contains buttons to access the Display Wells and Analyze Wells dialog boxes and the Restore Defaults function Figure 6 26 Display Wells Analyze Wells Restore Defaults Temperature Bar Threshold Bar Selected Well Peak Height 84 50 16 59 B02 1361 47 Fig 6 26 The Control Area
167. nenenes 64 6 5 5 Printing Results i reed i REA DRE DR RRRRXRTR EE iavaradacaeedlecvdeatenccadeates hgvanadada cas 65 6 6 Melt Curve and Melt Peak Charts sessseeeesesenenenenenenenennnnnnnnnennne nnns 65 6 6 1 Melt Curve and Melt Peak Chart M nu cccccccecececececececeeeeeeeeeeeeeeeeeeeeeeesaeaeaeeenenes 67 6 6 2 Delete Selected Peaks inrer iiti bea tak cda bet ace covavendagnetaes ct VENERE Da BARRE YR RA Re 67 6 6 3 Edit Melt Peak Begin End Temperature cccccccceecececeeececeeeeeeeeeeeeeeneeeeseeasaneenenes 67 6 6 4 Melt Curve Peak Control Area eeeeeseeseeee eene nennen nnne nn nnn nnns ar rni 68 6 6 5 Melt Curve Peak Spreadsheet sess eene nennen nnne nennen nn nnns 68 6 7 Allelic Discrimination Module For Multiplex Data Only eee 69 6 7 1 Allelic Discrimination PlOL i4 2 conieci rere aaa Da rau Recon aa 69 6 7 2 Allelic Discrimination Plot Menu cccccccececececececeeeeececeeeeeeeeeeneeeneecesesaeaeeeneneneneneees 71 6 7 3 Allelic Data SpreadSheet c sssscceecccecssssseseeeeeesesseesssaeeeeensessaeaesaeeseeseessanaaaseneenegs 71 6 7 4 Automatic Manual Call ccccccsssscccessssceceseeseeeeeessceeeseaeceeeseaueeesseaaeeeseaaeseesseeeessages 72 6 7 5 Man ak C lS esnie 73 6 7 6 Display Mode i oerte ai E E a neveu des 73 6 7 7 Vertical Threshold eee ed eerie tnodo Deed teo de e Poe na rer Eve eda aeo
168. nennen nnn nnn nnn nnns 49 6 2 2 Select Analysis MOd e 2 iur eese coc eese inier e te tr kae Co Dee ege e REC OR he Cue avari uY 50 6 2 3 Log VIEW BUON Ss ts 1i etre eee thinner rene er PR Ee EE tei aea edel ha oda Exe eta Reo Ri as 50 6 2 4 Selecting and Viewing Traces ccccececececeeeeeeeeeeeeeeeeeeeeneneeeneeeeeneneeensesaeaeneeseeneneass 51 6 3 Standard Curve Chart P 53 6 3 1 Standard Curve Chart Menu ccccccececececececeeeeeeeeeeeceeeeeeneesecneeeeeeeeeeeesaeaeeeeeeeneneaes 54 6 4 Results Sect ON c i iet etie coe ener Cena da EL R DR ELE A eR COR EERRR CERA ERR LDR ERA RH ia idaan 55 6 4 Plate Spreadsheet cct aaa aara apa Arv ea ERR ER Ens e aa Aanand Deea 55 6 4 2 Standard Curve C Results Spreadsheet eessesessseeeee nennen nnne nn nnn nnn 56 6 4 3 Amplification Data RFU Spreadsheet eseeseeseeeeeee nemen nnne 57 6 5 Amplification Plot Context Menu essen nennen nnn nn nnne nnn nn nnn nnn nnn nnne 57 6 5 1 Data Analysis Options oriin cer teen e epe natu lan eee een ex Le e EVEE vene Feed EVER deed EY us 57 6 5 2 Amplification Plot Data Display Options eseeeeeeeeneeeen nme nnnm 61 6 5 3 Data Export Options 2 1 echas er eaae uen CANKE AETI e ee a loo rasa ace yea dab geek a eR cigi RR DROIT 64 6 5 4 Exporting Results to Microsoft Excel ccccccccececececececeeeeeceeeeeeeeeeeeeeeeesaeenaeeeee
169. ng rear cover from the iCycler chassis by sliding the cover towards the front of the iCycler base 2 Install the modified sliding rear cover provided with the optical reaction block ensuring that the notch is oriented towards the rear of the iCycler thermal cycler Push the sliding rear cover on top of the chassis as far back as possible 4 Rotate the green latches on the optical reaction block up towards the open lid Lift the optical reaction block by the handle and install it onto the chassis Lower the front portion of the reaction block so that it engages with the chassis before the rear portion The rear of the block lid should fit over the front lip of the sliding rear cover 6 Secure the optical reaction block in place by rotating the green latches downward Close the optical lid Section 1 Getting Started 1 2 3 Installing the Support Bracket A support bracket with roller is provided for the MyiQ2 MyiQ or iQ5 optics module It is mounted to the rear of the iCycler thermal cycler Align the optics module support bracket with the two holes on the rear of the iCycler thermal cycler Using a 2 Phillips screwdriver adjust the height of the bracket with two of the appropriate screws provided with the system accessories Both of the screws should be approximately in the center of the slots on the bracket 1 2 4 Installing the Optics Module 1 Remove the plastic sheath and protective label from the optics module and place the
170. ns on the amplification chart determine which fluorophores appear in the standard curve chart shown below in Figure 6 7 The bottom of the chart displays a legend that includes The color used to plot each fluorophore The name of the fluorophore The efficiency of the reaction The coefficient of determination R 53 Section 6 Data Analysis Module e The slope of the line e The y intercept You can enlarge the standard curve chart by clicking on the plus sign in the upper left corner of the section aJ Standard Unknown 35 30 v 9 oO 25 B o E 20 d LT 15 10 0 2 4 6 8 10 Log Starting Quantity copy number e FAM E 95 0 R 220 999 slope 3 448 y int 39 e HEX E 96 5 RA2 1 000 slope 3 410 y int 39 Fig 6 7 The Standard Curve Chart 6 3 1 Standard Curve Chart Menu Right clicking on the standard curve chart opens a menu Figure 6 8 Copy Graph Print Data Print Graph Restore Graph Show Labels Fig 6 8 The Standard Curve Chart Menu This menu includes the following options e Copy Graph Copy Graph copies the standard curve chart to the clipboard To copy the entire graph enlarge the chart by clicking the button in the upper left corner of the standard curve Chart Then click Copy Graph in the menu 54 Section 6 Data Analysis Module e Print Data Print Data prints the Standard Curve C Results spreadsheet to your specified printer in Windows e
171. o fluorophores The fourth and fifth column present the RFU or threshold cycle data for both fluorophores and the last column is the genotype call made by either the software or the user 71 Section 6 Data Analysis Module Control WT Control WT 1109 2 3305 54 Control Control WT Control WT 841 64 3443 69 Control Control WT Control WT 527 05 2935 91 Control Control WT Control WT 384 4 2604 26 Control Control WT Control WT 2446 98 97 95 Control Control WT Control WT 2829 31 103 97 Contro Control WT Control WT 2862 07 42 02 Contro Control WT Control WT 2767 72 3 35 Contro NTC NTC 74 64 66 58 None NTC NTC 67 28 97 83 None NTC 54 89 142 04 None NTC NTC 151 4 36 8 None 1278 73 3751 75 Allele2 802 44 1860 93 Heterozygote a 1573 08 Heterozvaote Fig 6 30 The Allelic Data Spreadsheet In Automatic Call mode the iQ5 software creates the assignments based on the positions of the vertical and horizontal bars In Manual Call mode the Call column becomes editable through a menu This can be very useful if you want to change the definition of a sample from an unknown to a positive control for example The data in the spreadsheet may be copied to the clipboard for export to another program by clicking in the top left corner of the spreadsheet and typing CTRL C 6 7 4 Automatic Manual Call Select the type of analysis by clicking Automatic Call or Manual Call Figure 6 31 Q automatic Call Manual call
172. on 4 Gene a Ctrl Expression aa Mean Ct ct SD 1 OHrs Actin gt N A N A N A 15 82 0 01372 2 0Hrs Gapdh N A N A N A 18 21 0 02486 3 0Hrs ILib 1 00000 0 02790 0 02790 22 54 0 03871 4 0Hrs Tubulin 1 00000 0 01683 0 01683 19 90 0 02074 5 jiHr Actin N A N A N A 15 83 0 01933 6 1Hr Gapdh N A N A N A 18 16 0 05477 7 iHr ILib 0 22981 0 01090 0 01098 24 69 0 06384 g 1Hr Tubulin 3 88658 0 16308 0 16876 17 85 0 05558 9g 2Hrs Actin N A N A N A 15 77 0 02579 10 2Hrs Gapdh N A N A N A 18 05 0 01923 11 2Hrs ILib 0 05234 0 00148 0 00158 26 80 0 03853 12 2Hrs Tubulin 19 12670 2 01803 2 07211 15 38 0 15717 Fig 6 47 Gene Expression Analysis Data Table 89 Section 6 Data Analysis Module Corrected Values Calculations The efficiency value E used in gene expression calculations has an associated error If a standard curve was generated as part of the real time PCR assay this error can be calculated and used to adjust the error associated with the following standard deviation values e Rel Quant SD e Unscaled Expression SD e Expression SD Corrected Values for all gene expression results are displayed on the Data Table spreadsheet when Show Details is selected Figure 6 48 When Show Details is active three new columns appear in the Data Table spreadsheet The new columns display the error correction propagated from the standard curve data for e Relative Quantitation Standard Deviation e Unscaled Expression Standard Deviation
173. on Name entries have a character entry limit of 15 characters The Condition List option represents particular tests or treatments being evaluated for the purposes of your experiment A condition can be as simple as sample 1 or complex as mouse 123558 liver PMA though the latter example is often too long to present on a graph You can view conditions as samples in the Condition List spreadsheet as shown in Figure 6 44 MEN Setting HA DataTable cene List Condition List Data Set List Show Name Full Name Ctrl Color Graph 1 Std 3 Std 3 im D 0 Hrs 0 Hrs vi BEEZH Iv 3 Std 5 Std 5 rs az 73 4 Std 4 Std 4 l 5 1Hr 1Hr O E 6 Std 6 Std 6 C ag UC 7 Std 7 Std 7 ANN T 8 2Hrs 2Hrs a EN 9 Std 1 Std 1 m 10 Std 2 Std 2 O mE Fig 6 44 The Condition List 87 Section 6 Data Analysis Module Setting Control Conditions You can assign one condition as the control condition by selecting a checkbox next to the sample in the Condition List as shown in Figure 6 44 The iQ5 software assigns the control sample a value of 1 for every gene and all other conditions will be presented with values relative to this one This makes it simple to evaluate fold expression relative to the chosen control sample NOTE The control condition will have a value of 1 for all genes To access the condition list 1 Make sure the Settings radio button is activated 2 Click Condition List option 3 Select the
174. ondition names e Analysis method radio buttons Located above the gene expression plate interface section Used to select between normalized and relative quantity gene expression analysis methods PCR Quant EN Metcurve Peak M End Point Allelic Disc Gene Expr Edit Plate m Setting NEN Data Table ub ED Tubulin Qceneuist Condition List Data Set List 3 206 01 Show Auto Efficiency 1 60E 01 Name Full Name Ref Color Graph Efficiency 8 00E 00 Gene 1 Tubulin Tubulin r BE iv 95 0 5 4 006400 Y Axis Options 2 Actin Actin v EN iv 964 92 3 Lib Lb E Iv 96 8 amp gi eee Liner 4 Gapdh Gapdh v BEEN Iv 96 2 1 00E 00 Scaling Options A 5 006 01 Highest g s 2500 E Unsoed 1 25E 01 Graph error Saez StdDevs 3 13E 02 Ors 1Hr 2Hrs Condition Gene Name z Condition Name z Normalized expression ddat Recalculate HEB Copy conditionto all data sets MEM Enable for Gene Sudy Analyze Wells Relative quantity dct 1 2 3 4 5 6 7 8 g 10 u 12 A 19 93 17 79 15 46 Unknown Unknown Unknown 19 89 17 85 15 20 Unknown Unknown Unknown 19 90 17 90 15 49 eR Unknown Unknown Unknown Fig 6 38 The Gene Expression Analysis Tab Window 79 Section 6 Data Analysis Module Basic Workflow Steps for Gen
175. ow to remain open after the iQ5 software re analyzes the wells If you are satisfied with your selection click OK to close the dialog box and update the wells to include in the analysis If you click Cancel the iQ5 software closes the dialog box and discards all changes NOTE This procedure does not permanently remove data This procedure only removes that data from the current analysis You can add the excluded wells back to the analysis at any time by including them in the Select Wells to Analyze dialog box Display Wells Click the Display Wells button on the PCR Quant screen to select the wells that you wish to include in the data display Display Wells After you click Display Wells the Select Analyzed Wells for Display floating window appears as shown below in Figure 6 6 You can include or exclude wells for display in the Select Analyzed Wells for Display floating window You can only choose from analyzed wells Wells that have been excluded from analysis in the Select Wells to Analyze window do not appear in the Select Analyzed Wells for Display window Selecting wells for display does not change the underlying data analysis Therefore the calculation of thresholds and replicate statistics does not change 52 Section 6 Data Analysis Module Wa Select Analyzed Wells for Display AUNI AOI aaa
176. ples in the graph You may zoom in on the allelic discrimination plot by first selecting Zoom Enabled from the plot menu then left clicking and dragging over the area you wish to enlarge Zoom out by choosing Restore Graph from the context menu 6 7 2 Allelic Discrimination Plot Menu Open the allelic discrimination plot menu by right clicking on the allelic discrimination plot Figure 6 29 Adjust Graph Restore Graph Zoom Enable Show Labels Copy Graph Print Graph Print Data Fig 6 29 The Allelic Discrimination Plot Menu e Adjust Graph This feature allows you to change the way that the plot is presented The maximum and minimum values for either axis may be entered directly into the text boxes Alternatively the up and down arrows can be used to set the maximum and minimum values e Restore Graph Use this to redraw the graph after zooming e Zoom Enable Use this to enlarge the desired plot area e Show Labels Labels each sample on the Allelic Discrimination plot with its well name e Copy Graph This will copy the displayed Allelic Discrimination plot to the clipboard for import into other programs e Print Graph This prints only the graph e Print Data This prints the Allelic Discrimination spreadsheet data 6 7 3 Allelic Data Spreadsheet A six column spreadsheet is displayed in the window Figure 6 30 The first column is the well number and the second and third are the identifier entered at plate setup for the tw
177. port Templates Lr Template RMEData TemplateFAM_HEX C3 SampleFiles N ub 3 SupportFiles Users admis Run Time aa ommy Central Jii ix mS Selected Protoool 2Stepimo z TemplateFAM HEXpts A E Create New Protocol f Edit 1 Create Ni Plat T dm ee _Create New ERE Original O Current Original O Current SLi Vessel Type Plates Data Analysis Ao E 5S o6 O00 Calibration Tah a PCR Melt Data Acquisition User Profile eee 0e0e0800 z 00000000 550 B9 RealTime C E UC 006020 Fig 4 1 The Setup tab 19 Section 4 Workshop Module 4 1 1 File Browser Section Located in the top left of the Setup window the file browser area contains a folder tree on the left side and a file list on the right side The files displayed in the list depend upon the selected Setup window tab With the Setup tab active the displayed Setup window contains four additional tabs e Protocol Used to select edit or create a protocol for running a real time experiment If the Protocol tab is selected the iQ5 software displays protocol files file extension tmo The selected protocol appears in the Selected Protocol window e Plate Used to select edit or create a plate setup for running a real time experiment If the Plate tab is selected the iQ5 software displays plate setup files file extension pts The selected plate appears in the Selected Plate Setup window e Run Set Consists of a linked
178. quation listed above to calculate normalized expression If a control is assigned relative quantities for all genes within the control sample are equal to 1 This results in normalized expression of 1 for the control and relative quantities for all other conditions will be presented relative to this normalized control sample According to the calculations performed by the iQ5 software normalized expression is equivalent to unscaled normalized expression analysis when a control is chosen Normalization Factor The denominator of the normalized expression equation is referred to as the normalization factor The normalization factor is the geometric mean of the relative quantities of all the reference genes for a given sample Normalization Factor sample ene Rel Quant Rel Quant au ern Rel Quant a petm sample Ref 1 Scaled Normalized Expression Scaling to highest or lowest is achieved simply by dividing all unscaled normalized expression values for a given gene by the highest or lowest expresser respectively Scaled to Highest Normalized Expression annie gene v Scaled Normalized Expression sampie tgene Normalized Expression s tgene x Scaled to Lowest Normalized Expression sempre tgene x Scaled Normalized Expression sampe gene x Normalized Expression s tgene 9 96 Section 6 Data Analysis Module Standard Deviation for the Normalized Expression Rescaling this value is accomplished by
179. quations used in determining the error propagation are the Standard Error Standard Error for Normalized Expression and the Standard Error for the Normalized Gene of Interest GOI The equation for Standard Error is shown below SE als 90 Section 6 Data Analysis Module Where 7 number of measurements The Standard Error for Normalized Expression equation is shown below 2 2 2 Sere MEE SE Rel Quant wmpeRef SE Rel Quant smpeRefz o SE Rel Quant sample Re f n nXRel Quant ample Re f 1 nxRel Quant ampie re 2 nXRel Quant sample Refn Where VF Normalization Factor The Standard Error for Normalized GOI equation is the following 2 2 SE GOI norm z GOL sorm x NP BE ad NF GOI Where GOf j44 the Normalized Gene of Interest Exporting Data from the Data Table 1 Make sure the Data Table button is activated If not click Data Table 2 Right click on the spreadsheet A shortcut menu appears Figure 6 49 3 Select Export to Excel 4 Enter a name and file destination for the Excel file generated with your data export EN Data Table Fig 6 49 Exporting Data Table Spreadsheets to Microsoft Excel Printing Data The Print command on the spreadsheet menu will print the displayed spreadsheet Gene List Condition List Data Set List or Data Table When selected a Print Preview box is opened which contains an illustration of the spreadsheet as it will appear once printed Click
180. r by clicking Display Data in the amplification chart menu When you click Print Std Curve Data the Print Preview dialog box appears so you can print the amplification data NOTE If you alter the width of the columns in the spreadsheet in the Standard Curve C Results spreadsheet the printed spreadsheet will reflect that change 6 5 4 Exporting Results to Microsoft Excel Exporting Data from the Results Table 1 2 3 4 Make sure the Results button is activated If not click Results Right click on the spreadsheet A shortcut menu appears Figure 6 21 Select Export to Excel Enter a name and file destination for the Excel file generated with your data export 64 Section 6 Data Analysis Module Well Fluor Identifier cJ Replicate Threshold Cycle ct 19 93 17 79 15 46 19 89 17 85 15 20 19 90 17 90 15 49 11 63 15 53 19 02 0 Hrs 1Hr 2 Hrs 0 Hrs lHr 31 B04 32 B05 33 B06 34 C04 35 C05 36 C06 37 D04 38 D05 39 D06 40 F03 41 F04 42 F05 rs Export to Excel jrs Unkn 1 Hr Unkn 2 Hrs Std Std Std U Ne UU MN e WN m 0 N ne Fig 6 21 Exporting PCR Quant Data Tables to Microsoft Excel The Export to Excel command is useful for exporting exact values from the spreadsheet When the Export to Excel command is selected from the menu an Export to Excel file save box is displayed Choose a location where the Excel file is to be saved and click Save The iQ5 software autom
181. r decrease as frequently as every cycle and the increase or decrease can begin following any cycle The time increment or decrement may be as large as desired as long it does not result in dwell times which are outside the limits of 00 00 and 99 59 To program a time increment or decrement 1 Click Time Change in the Show Options box Three new columns will appear in the spreadsheet 2 Forthe repeated step that you want to affect enter the change desired in the Time Change column To decrement the time enter the decremental change as a negative number for example 0 05 3 Enter the cycle in which you want the change to occur for the first time in the Begin Repeat column Usually it is cycle 2 but it can be any cycle greater than 1 4 Enter the frequency that you want the change to occur in the How Often column Usually you will want the change to occur every repeat so enter 1 in this column Cycle Description Step Process You can choose from a list of descriptive names or enter one of your own to describe cycle or step processes A cycle description or step process may be entered in the spreadsheet in the following manner 1 Click Cycle Description or Step Process in the Show Options box 2 Click the cell of the spreadsheet for the cycle or step you wish to name and either choose one of the listed names from the pull down menu or enter one of your own 34 Section 4 Workshop Module 4 3 4 Gradient A thermal gra
182. r sample you define Next 1 Click Apply Plate Changes to make the changes To see the effect on analysis go to one of the other Data Analysis windows Apply Plate Changes For more information about Edit Plate functions refer to section 4 2 6 12 The Reports Tool The Reports tool of the iQ5 software is accessible from the menu bar of the iQ5 software Clicking on the heading for Reports will open the Report Viewer Figure 6 64 The Reports Viewer pulls data charts and graphs directly from the displayed settings of the Data Analysis module currently in use For this reason it is best to complete all desired analysis and formatting of the displayed data prior to using the Reports tool a Report Viewer Gene Expression Reports No report file selected Select Report None X Sort Data By None Ascending Order Descending Order Page Setup Print Preview Print Save to File Fig 6 64 The Report Viewer Window for Creating New Reports 107 Section 6 Data Analysis Module 6 12 1 Report Viewer Options The following options are available from within the Report Viewer window e Select Report Use this drop down menu to select the level of detail in the report for your dataset The number and type of reports available will vary according to the Data Analysis Module currently being used e Sort Data By Within a given report all data displayed in tables can be sorted by
183. r thermal cycler 96 well optical reaction block MyiQ2 optics module iQ5 system software on CD ROM optical quality 96 well PCR plates sealing tape communication cables system accessories power cords instructions 96 well Thin Wall PCR Plates 25 per box Hard Shell Full Height 96 Well Semi Skirted PCR Plates Optical Quality Sealing Tape 100 sheets 0 2 ml 8 Tube Strips Without Caps Optical Flat 8 Cap Strips Replacement Halogen Lamp iQ5 Software Installation Disk MyiQ2 Calibrator Dye Solution Kit 1x calibration dye solutions 3 tubes each of FAM HEX TET Joe External Well Factor Solution 5 tubes Fluorescein Calibration Dye iQ Supermix 100 x 50 ul reactions 2x mix iQ Supermix 500 x 50 ul reactions 2x mix iQ SYBR Green Supermix 100 x 50 ul reactions 2x mix iQ SYBR Green Supermix 500 x 50 ul reactions 2x mix iScript CDNA Synthesis Kit 25 x 20 ul reactions includes 5x iScript reaction mix iScript enzyme nuclease free water iScript CDNA Synthesis Kit 100 x 20 ul reactions iScript Select cDNA Synthesis Kit 25 x 20 ul reactions includes 5x iScript Select reaction mix iScript reverse transcriptase oligo dT mix random primer mix gene specific primer GSP enhancer solution nuclease free water iScript Select cDNA Synthesis Kit 100 x 20 ul reactions iScript OneStep RTPCR Kit With SYBR Green 50 x 50 ul reactions iScript OneStep RTPCR Kit With SYBR Green 200 x 50 ul reactions iScript OneStep RTP
184. r unknown samples ertical Threshold 1266 74 Fig 6 35 The Vertical Threshold Box 6 7 8 Horizontal Threshold The Horizontal Threshold box Figure 6 36 displays the current position of the horizontal bar This box is not editable The position of the horizontal bar along with the position of the vertical bar determines the genotype assignment of unknown samples Horizontal Threshold z 1415 96 Fig 6 36 The Horizontal Threshold Box 6 7 9 Normalize Data When the RFU option button is selected the Normalize Data option Figure 6 37 appears as shown below if the plate setup contains a no template control NTC sample type for both Allele 1 and Allele 2 EA Normalize Data Fig 6 37 The Normalize Data Button The RFU data may be normalized and displayed on a plot that ranges from 0 to 1 on both axes This display is sometimes a very effective presentation of RFU data The RFU data is normalized to the NTC value as a linear combination of Allele 1 and Allele 2 specific signals The iQ5 software uses the following formula initially introduced by Livak et al 1995 Al Normalized Az A Az X NTCa a2 74 Section 6 Data Analysis Module Where A represents Allele 1 A represents Allele 2 X represents the mean NTC 41442 represents the sum of NTC for Allele 1 and Allele 2 Reference Livak JL Marmaro J and Todd JA Towards fully automated genom
185. ration page 111 o Preparing calibration plates page 112 o Performing calibrations page 112 o Viewing calibration files page 119 o Troubleshooting optics with the Image Window page 120 7 1 Calibration Overview To run experiments on the MyiQ2 MyiQ and iQ5 systems requires that the instruments are calibrated There are four different calibrations required for the MyiQ2 and iQ5 systems mask alignment background calibration persistent well factor generation and pure dye calibration NOTE Pure dye calibration is NOT performed on the MyiQ Real Time PCR Detection System Calibration should be performed when e The instrument is installed e Any of the components that affect the light path are replaced such as the lamp filters optical lid or optics module e Whenever prompted by the software as calibrations expire Calibration factors are specific for the optical path you are using which includes the reaction vessel and sealing mechanism used in the experiment you wish to run Consequently calibrate using the reaction vessel type plates or tubes and sealing method film or caps you will use for your future experiments The iQ5 software requires persistent well factor calibration for all combinations of reaction vessel sealing product and reaction volume that you anticipate using even if dynamic well factor collection is selected This feature prevents the run aborting if dynamic well factor collection from the experimenta
186. ress is monitored in the top left hand section of the window NEN Init Run Mm Show Plate gm Monitor Run Data File Data 2008 11 11 1033 0pd Selected Protocol 2Steptmo Pause Run DwellTime TB Current Protocol State Run Time MBB cutie Protocol Exiting __Pause Stop Run_ 00 10 Ia ise cde Start 1033 NEEE ining 00 10 A Step 1 Stop est 10 33 Temperature Cebi Repe 26 of 40 Con SetPoint 95 0 Run State Running be Running user selected Protocoland Plate in Emulation Mode Running Sample 81 4 Step State Ramping 4 PCR Quantification Selected Plate Setup Hex FAMpts E 11000 E i Sample Volume 25ul Seal Type Film Vessel Type Plates E f 10000 x E ox on jJ p s000 1 EN 00309 516r ior mmm B s000 A BG 3 WC C2 NU a tt et BEEN S g Z 7000 s PRESSE A a E f Pe EEQOOUUCUEEan e E e SE L H il IIo I A a F amp ee 000 F rH r E EH 5 Yrs Wes WO MI 5 IM 5 Bane lt a joooung i s BMELIXJICLCIPCIPIBNSSN 0 10 20 30 B EBDCIXJLISSEE Display Selected Wells Fig 5 5 The Monitor Run Window 5 7 Run Time Protocol Editing During a run you can access real time pro
187. ression Example 3 RMEDate ar MyiQ Dynamic Range Data FAM Notes 7 Artificial Time course in which SampleFil 4r MyiQ End Point Fluorescein Dilution Data SYBR 1 Actin is constant at 1e5 cps rxn C3 Supporti a MyiQ Gene Expression SYBR Data SYBR2 Gapdh is constant at 1e6 cps rxn A 2 SYBR3 IL1b decreases 4 fold with time Mu cna SIDE DOC SYBR4 Tubulin increases 4 fold with time Fig 5 1 Accessing the Run Time Central Module 5 1 Initiate Run Window Run Selected Use the Initiate Run window Figure 5 2 to confirm run conditions and then to initiate the optical data run The protocol to be run is in the bottom left corner of the window and the plate setup that will be used appears in the bottom right corner The type of well factors to be used in the run can be selected in the top left section of the window Record details of the experiment in the Notes box These notes will be incorporated into the experimental file To begin a run select your desired well factor collection method and click Begin Run The Save dialog box opens Type a unique name for the optical data file The iQ5 software saves data automatically during the experiment 41 Section 5 Run Time Central Module Ente run MN 7 Shwrus MN 0 Monitor Run __ Use Persistent Well Factors Notes Collect Well Factors from Experimental Plate Running user selected Protocol and Plate Begin Run S
188. rform pure dye calibration as described in section 7 4 4 7 4 4 um Mask EH Background EM wWellFactors um Pure Dye Instructions Referto Help Fileforinstructions on howto run this type of calibration Seal Type Film Le Le Vessel Type Plates Collect Persistent Well Factors Fig 7 4 The Well Factors Window Performing Pure Dye Calibration Before you perform a pure dye calibration run complete the following steps 1 aa FW DN Prepare a pure dye calibration plate see section 7 3 Ensure that the mask has been aligned Ensure that background calibration has been performed Ensure that persistent well factors have been generated Place the pure dye calibration plate in the iCycler thermal cycler Select the Pure Dye tab in the Calibration module The Pure Dye Calibration window is shown in Figure 7 5 116 Section 7 Calibrating the Instrument To perform a pure dye calibration run 1 Select from the Pure Dye Plate Setup file selector window or click Create New Ensure appropriate dyes are selected that are compatible with the filter setup of your connected instrument If incompatible dyes are selected the following error will appear Bio Rad iQ5 Qo Plate contains fluorophore s that cannot be used with the current instrument Show error details 2 Click Run Pure Dye Calibration 3 When the iQ5 software completes the pure dye calibratio
189. rrent cycle Insert a step Insert a step by clicking in the Insert column within a step row Steps have a white background The iQ5 software inserts the new step below the current cycle You can use the Options cell to customize whether the step or cycle is inserted before or after the current cycle step as well as how many steps the iQ5 software will insert when you insert a cycle Delete a cycle Delete a cycle by clicking in the Delete column within a cycle row Cycles are indicated with a blue background Delete a step Delete a step by clicking in the Delete column within a step row Steps are indicated with a white background 2 Save the protocol by clicking Save amp Exit Protocol Editing Enter the name of the protocol in the Save As dialog box and then click Save 2 2 Plate Setup Quick Guide Within the Workshop module 1 Open the Plate Setup Editor Window using one of the following methods Click Create New in the plate setup display pane to enter the Plate Setup Editor or Click Plate and select the desired plate setup file from the file tree directory Double click the file name to go directly to the Plate Setup Editor or Click Plate and select the desired plate setup file from the file tree directory Click the file name to open the plate setup in the bottom right section of the Workshop TE Section 2 Quick Guides window Click Edit to open the plate setup in the Plate Setup Editor or e Click Data File and s
190. rs select Search for the best driver in these locations and then click the Include this location in the search checkbox Use the browse button to navigate to the iQ5 Drivers folder The default location of the drivers is C Program Files Bio Rad iQ5 Drivers Click Next to continue Next Windows will display a Hardware Installation dialog box regarding Windows Logo Testing a service offered by Microsoft To complete driver installation for the iQ5 or MyiQ systems click Continue Anyway 1 4 Recommended Computer Settings For the MyiQ2 MyiQ and iQ5 systems to communicate properly with the iQ5 Optical System software version 2 1 the computer settings should be set as described below Section 1 Getting Started Computer Power Management Settings on Windows Vista 1 From the Start menu choose Control Panel Switch the Control Panel view to Classic View Choose Power options and then choose Create a Power Plan Choose the High Performance plan then enter the Plan name iQ5 software and press Next Change the Turn Off The Display and Put the Computer to Sleep options to Never Click Save Changes Select your new iQ5 software power plan by clicking on the radio button open Change Advanced Power Settings to set the following conditions a Adjust settings to turn off hard disk after 180 minutes b For Sleep Settings i Set Sleep After setting to Never ii Set Hybrid Sleep setting to On iii Set Hib
191. rument 7 4 5 Editing and Creating Pure Dye Plate Setups Selecting a Pure Dye Plate Setup Select a Pure Dye Plate Setup from the file selector window within the Pure Dye Calibration tab When loaded the fluorophores on the plate will display within the plate display in the lower right hand corner of the Pure Dye Calibration tab Check the Seal Type and Vessel Type fields If these fields match the intended procedure and the correct fluorophores are displayed proceed with pure dye calibration If any of these particulars vary from the intended experiment parameters the Pure Dye Plate Setup must be edited or a new Pure Dye Plate Setup created An example of a Pure Dye Calibration Plate Setup window is shown in Figure 7 6 Selected Plate Setup MyiQ2 Puredye calibrationpts Edit Create New Sample Volume 25ul Seal Type Film Vessel Type Plates om e ow je a e E x TT E e r2 9 9 9 9 G 9 Q Fig 7 6 Pure Dye Calibration Plate Setup Window Editing or Creating a Pure Dye Plate Setup To edit a Pure Dye Plate Setup select the Pure Dye Plate Setup that most closely resembles the experimental parameters desired and click Edit The Pure Dye Plate Setup editor will load as shown in Figure 7 7 Jm ose JUN _ background ON Wel radors mm Pure Dye _ Editing Plate MviQ2 Puredye calibration pts Sample Volume ps Save amp Exit Plate Editing Nos Selas Fin 7 Cancel amp Exit Plate Editing
192. run Calibration Errors When opd files are merged into a Gene Study that does not meet the above criteria a warning message will be displayed which states that the genes do not contain common samples across all data sets Figure 6 62 The warning message will specifically list the genes that are in violation By clicking the warning message OK button the analysis will proceed without any inter run calibrators Clicking on the Inter run Calibration button when there are no calibrators assigned will display a message box which states that inter run calibrators are not available Proceeding without inter run calibrators may cause inaccurate representation of the data To correct this problem change the Gene and or Condition Names in the plate interface so that identical Gene and Condition Names are used across all data sets of the Gene Study Use the Gene Name and Condition Name drop down menus to select the appropriate Gene and Condition Names or directly input the correct Gene and Condition Names To change a large number of Gene and Condition well names across several plates use the Copy and Paste command from the right mouse button menu x The following genes do not contain common samples across all data sets Tubulin gapdh Data for these genes will be presented with no inter run calibration and may be inaccurate Double check your gene names and condition labels to make sure that they are consistent across all plates Fig 6 62 No Inter
193. run editing of the experimental plate setup allowing you to correct erroneous sample type assignments The Data Analysis module is opened automatically when you open a saved data file from the Workshop Calibration Module In order to extract the best data from your real time PCR experiment the MyiQ2 MyiQ or iQ5 systems must be calibrated These simple and easily performed calibration routines are accessed in the Calibration module There are calibration routines for Pure Dyes Mask Alignment Background and Well Factors collection User Profile Module This module can be used by the Administrator to add new users and to set users access restrictions to various functions of the iQ5 software All users can use this module to set their personal preferences for the iQ5 software 18 Section 4 Workshop Module Section 4 Workshop Module This section contains information on the following topics o Setup Tab page 19 o Plate Setup page 21 o Protocol page 31 o Run Set pg 37 o Opening a Data File page 39 o Applying Alternate Calibration Files page 39 The Workshop module consists of the Setup and Plate Summary tabs For detailed information on the information contained in the Plate Summary tab refer to section 4 2 10 4 1 Setup Tab The Setup tab window Figure 4 1 consists of four sections File View Repo Tools Help Masks n puredyestarter Persistence Sample files Workshop Re
194. s then no genotype can be assigned Genotype assignments for unknown samples may also be determined by plotting the threshold cycle for one fluorophore allele 1 on the x axis against the threshold cycle for the other fluorophore allele 2 on the y axis on the allelic discrimination plot If the unknown threshold cycle values are greater than the horizontal and vertical bars then the genotype is none or not assigned Samples not crossing threshold during the protocol are placed in this quadrant If the unknown threshold cycle values are greater than the horizontal bar and less than the vertical bar the genotype is homozygous for allele 1 allele 1 threshold cycle is plotted on the x axis If the unknown threshold cycle values are less than the horizontal bar and greater than the vertical bar the genotype is homozygous for allele 2 allele 2 threshold cycle is plotted on the y axis If the unknown threshold cycle values are less than the horizontal and vertical bars then the genotype is heterozygous 70 Section 6 Data Analysis Module In Manual Call mode the plot displays the RFU or threshold cycle data only The threshold bars do not appear and modifications to calls are made manually by using the drop down menu in the Call column of the data spreadsheet Alternatively modifications to calls are made by choosing the call type from the radio buttons in the Allele Call box and then clicking and dragging to select desired sam
195. s for four major settings that will impact the analysis or display of data collected from your real time PCR detection system PCR Quant Baselining Auto Calculated User Defined User Defined Baseline Cycle Range f EZ to 24 Log View on eot Analysis Mode PCR Base Line Subtracted Curve Fit v Grid Rows Em Identifier E Ct E Concentration Em EP Call Fig 8 6 Defining User Specific PCR Quant Analysis and Display Options e Baselining By default PCR baselines are auto calculated With the Baselining user preference setting you can override auto calculation by clicking User Defined After you dick User Defined the iQ5 software activates the User Defined Baseline Cycle Range boxes Enter the desired values for the start cycle and ending cycle for all traces Auto Calculated is the factory default Log View Use this option to select either a semi logarithmic or linear view of the PCR amplification chart data When the Log View option is On a semi logarithmic view of the amplification data will be displayed The factory default for Log View is Off Analysis Mode You can select from three analysis modes in the Analysis Mode drop down list boxes The three analysis modes include Background Subtracted PCR Base Line Subtracted PCR Base Line Subtracted Curve Fit PCR Base Line Subtracted Curve Fit is the factory default e Grid Rows The Grid Rows options are a set of checkboxes that you can use to display additional deta
196. sheet 8 3 Logging on to the iQ5 Software There are two ways for a user to sign in to the iQ5 software e At the start up of the iQ5 program e Through the Switching Users option in the Tools menu EEn Fig 8 11 The User Login Dialog Box 1 The user name can be selected from the pull down menu in the Login dialog box Figure 8 11 Enter the password and click OK to log on to the iQ5 software 2 If the user name or password is incorrect an error message will be displayed Figure 8 12 Login failed x Make sure you have the correct password and user name taking care to type the password correctly including capitalization Make sure the Caps Lock has not been accidentally turned on Fig 8 12 Incorrect Login Error Message 130 Section 8 User Profiles 3 Click OK and enter the correct information If you have forgotten your password it may be reset by the iQ5 Administrator 4 The current user can be determined by looking at title bar in the top right corner of the software The current user name will be displayed in parentheses next to the Bio Rad iQ5 header Figure 8 13 Wa Bio Rad iQ5 Russell Lab a ReportTe or 2Step c3 RMEDat Sample files Workshop C SampleFil M SunnartFi Fig 8 13 User Name Displayed in the iQ5 Software Title Bar 8 3 1 Switching Users To switch a user 1 Click on the Workshop module icon then select Switch User from the Tools menu 2 The
197. sion analysis Gene Expression Relativeto Zero Control XAxis Condition Gene YAxis Log2 Linea Scaling Highest Lowest Unscled Method ddct dc Std Devs31 00 Fig 8 9 Defining Analysis and Display Options for the Gene Expression Module Relative to This option allows you to present data with axes originating at 1 relative to control or at zero relative to zero X Axis This option allows you to graph either genes or conditions on the x axis Y Axis This option allows you to display the graph with the y axis in a log2 scale or on a linear scale Scaling Scale to control is another option which is accomplished by assigning a control in the Condition List Method These options allow you to set the default analysis mode for your gene expression analysis You can select ddCt for normalized expression or dCt for relative expression as a default setting Std Devs The default presentation for the error bars is plus and minus one standard deviation You can change the multiplier to get plus and minus 2 or 3 standard deviations 8 2 User Administration The Administration tab can be used by a user with an iQ5 Administrator role to Add or delete users Edit user details that is full name and Email information Set or change passwords Control the permissions of each user Only an iQ5 Administrator can access and modify information contained in the Administration window Users specified as
198. software automatically calculates the threshold You can override auto calculation in one of two ways On the amplification chart move the cursor over the displayed threshold line until the cursor icon becomes a hand Left click on the displayed threshold line and drag the threshold to the desired position From the Base Line Threshold Parameter dialog box select User Defined in the Crossing Threshold section at the bottom of the dialog box Enter the desired value for the threshold position and then click OK 6 5 2 Amplification Plot Data Display Options There are seven additional data analysis options in the amplification plot menu Single Point All Candidates Adjust Graph Define Trace Style Display Data Restore Graph Show All Traces Single Point The Single Point mode of data presentation in which the iQ5 software averages all data collected at a particular step and then plots the average is the default mode For example if the iQ5 software collects four data points during the third repeat of an amplification cycle the mean of those 4 points is plotted at cycle 3 The alternative to viewing the data in Single Point mode is to view the data in All Candidates mode All Candidates In All Candidates mode the iQ5 software plots every single data point that is collected You cannot use automated data analysis features when the data are presented in All Candidates mode This mode is unavailable when the analysis mode is PCR Base
199. t Tolerance End Point Tolerance defines the margins for sorting unknowns as positives or negatives How the tolerance variable and the type of tolerance are applied depends on which method is selected The End Point Tolerance drop down list box consists of two choices e RFUs This is the default Tolerance choice and should be selected if you would like to use an absolute RFU value for the tolerance value The minimum RFU tolerance value is 2 whereas the maximum is the absolute value of the highest RFU value minus the absolute value of the lowest RFU value The default RFU tolerance value is 10 of the total RFU range The definition of the RFU range is dependent on the method chosen e For the negative method the range is the highest RFU value minus the negative control average e For the positive method the range is the positive control average minus the lowest RFU value e For the positives amp negatives method the range is the positive control average minus the Negative control average e Percent of Range Select this setting if you would like to use a percentage of the RFU range for the tolerance value The minimum percent of range is 1 percent whereas the maximum percent of range is 99 percent The default percent of range tolerance is 10 percent End Cycles to Average End Cycles to Average is the number of cycles from the last cycle which will be used to calculate an average End Point RFU value The End Cycles to Average fiel
200. te Setup Editor window This spreadsheet displays information about all wells simultaneously one fluorophore at a time Fluorophore selection is made at the top of the spreadsheet SYERT Import Identifiers Eat Plate Spreadsheet Edtng Rape Idertifer Ccnd tice Qsertty Units al Ba Rad 153677 oy eunte o Rd SE rad EME Rad SES Rad EDE LES SS SE SE eee ee eee eee PPUPPTPEPPTPTETPTETT D E J A i 1 8 Hi 1 ml 002 Fig 4 11 The Plate Editor Spreadsheet Within this spreadsheet you may change the sample type identifier and quantity Note that changes made to one member of a replicate group are carried through to all members of the 29 Section 4 Workshop Module replicate group Replicate group assignment changes should be made on the plate not in the spreadsheets This single dye layer spreadsheet has another feature that differentiates it from the single well spreadsheet From this spreadsheet you may Import Well Identifiers from an external comma separated values CSV file Importing Well Identifiers From the Plate Editor spreadsheet you may import well sample identifiers from an external CSV file The number of identifiers must match the number of wells before the import can be carried out The simplest way to use the import feature is to fill out the Identifier template file provided with the software in the sample files folder and then save the file in the CSV format 4 2 10 Plate Su
201. ted trace If you click the fluorophore selector buttons from the amplification chart traces from other fluorophores in the selected well also appear Viewing all traces To restore a view of all traces right click on the amplification chart and select Show All Traces Zoom To zoom in on a section of the plot click and drag with the mouse on the desired region To zoom out select the plot and then type R or right click on the plot and then click Restore Graph in the menu Analyze Wells Click Analyze Wells on the PCR Quant screen to select the wells that you wish to include in data analysis The Select Wells to Analyze floating window appears as shown in Figure 6 5 Analyze Wells F Select Wells to Analyze 8 9 10 11 12 CESICOHCSICNCIC AIC TONG DO 5 o oo oo Do oo I AAN Selected wells arehighlighted in yellow black borders Seled Deseled modifies wells inall dye layers SelectAll OK Cancel Apply Fig 6 5 The Select Wells to Analyze Window You can include or exclude wells for analysis in this floating window The original data are always preserved and excluded wells can always be added back to the data analysis in this window 51 Section 6 Data Analysis Module Removing wells from data analysis changes the calculation of the threshold location Changing the calculation of the threshold location may result in a change in the threshold crossing
202. tep in the Range cell of the spreadsheet The Gradient Display table Figure 4 15 will update with the temperatures at each row 4 You can change the range in the spreadsheet or make a direct entry of the range in the gradient display Press Enter and the display will update with the new calculated temperature for each row Fig 4 15 Gradient Display Table 35 Section 4 Workshop Module Programming a Specific Temperature for a Specific Row To program a specific temperature in any single row 1 Click Gradient in the Show Options box Two columns will appear in the spreadsheet and a representation of the gradient will appear on the right side of the window Click the Gradient checkbox in the spreadsheet for the desired step Enter the desired temperature into a specific single row on the gradient display Press Enter The temperatures for the other rows will be calculated based on the input desired temperature and the range NOTE You cannot specify the exact temperature on more than one row at a time S BUM 4 3 5 Melt Curve Peak Melt Curve Peak analysis is a dynamic tool used to measure the melting temperature Tm of double stranded DNA dsDNA molecules DNA duplexes can be visualized by either incorporation of DNA binding dyes for example SYBR Green I or by hybridization with fluorescently labeled probes In the case of DNA binding dyes and non cleavable hybridization probes fluorescence is brightest when t
203. the Save Optical Data File dialog box and then click Save End Point Analysis of an Newly Completed End Point Run 1 2 Once the real time PCR detection system completes the run the End Point tab is displayed Make selections for the following parameters e Method Use Negatives to differentiate samples that do not amplify the target sequence from those that do amplify the target sequence e End Point Tolerance and Tolerance Parameter 11 6 Section 2 Quick Guides Select the wells to analyze by clicking Analyze Wells Define the positive and or negative controls in the Define Controls column within the End Point Analysis table Click Recalculate The End Point Analysis table displays a positive negative or blank label for each unknown under the Unknowns Call column Click Reports to obtain customized reports of the End Point Analysis End Point Analysis of an Existing Data File 1 3 4 8 Within the Workshop module click Data and select your desired data file using the file tree browser Click Analyze Click the End Point tab Make selections for the following parameters e Method e End Point Tolerance and Tolerance Parameter Select the wells to analyze by checking Analyze Wells Click on Analyze Selected Wells and close the Select Wells for Analysis floating window when finished Define the positive and or negative controls in the Define Controls column of the end point analysis table
204. tion Instructions The filters designed for use in the MyiQ2 MyiQ and iQ5 optics modules are made of glass and mounted in plastic holders The filter holders are held in either the excitation or emission filter wheel of the optics module Each filter wheel holds six filters Every position in a filter wheel must have a filter or an opaque filter blank to avoid damage to the CCD detector The first position in each filter wheel is designated as the home position and must always contain an opaque filter blank Filters can be removed for cleaning or replacement If a filter shatters or breaks during the installation process contact your local Bio Rad office immediately for service do not attempt to remove the broken components from the interior of the camera housing It is critically important that the excitation and emission filters are in the correct positions in the filter wheels Please confirm that the filters are in the proper location after cleaning or replacing filters in the optics module Section 9 2 1 summarizes the positions of the filter pairs for the systems optical characteristics and the recommended fluorophores with which the filters are compatible 9 2 1 Recommended Fluorophores and Filter Specifications Recommended fluorophores for the MyiQ2 Real Time PCR Detection System e Filter position 2 485 30X 530 30M Fluorescein FAM SYBR Green I e Filter position 3 530 30X 575 20M HEX TET VIC JOE Recommended fluorophores for the My
205. tion Logs Data File Log Data File Info Calibration Data Analysis Parameters Fig 7 9 The View Menu Similarly the contents of the background and external well factor calibration files can also be accessed in this manner 7 6 Troubleshooting Optics with the Mask Image Window 7 6 1 Filter Position The Filter Position radio buttons Figure 7 10 are used to position the filter wheels The exposure time can be changed from this screen and an exposure taken Home locates the filter wheel to the blank position Refer to section 9 2 1 for information about system filter specifications and recommended fluorophores Filter Position Blak o Home Exposure Time ms 2048 x Take an Exposure Fig 7 10 The Filter Selection Frame for a MyiQ2 System 7 6 2 Camera Status Serial Number EP0021 FW version 1 032 Library version 1 026 Modd IQ2COLOR Lamp Time Warmup Time Fig 7 11 The Camera Status Screen 120 Section 7 Calibrating the Instrument The Camera Status screen Figure 7 11 provides feedback about the camera In this screen you can determine if The camera is connected The optical lid is closed The serial number firmware version library version and model of the camera The length of time the camera lamp has been in use since it was installed The length of time the lamp has been on since the camera was last turned on referred to as Warmup Time 121
206. tion using default begin and end temperatures and the default position of the threshold bar so data are not affected by edits to any of those parameters e Edited Begin Temp When the peak begin temperature bar is dragged to a new position it is reflected here e Edited End Temp When the peak end temperature bar is dragged to a new position it is reflected here 6 7 Allelic Discrimination Module For Multiplex Data Only The Allelic Discrimination module is useful for assigning genotypes to unknown samples by making comparisons to known genotypes It can be used to distinguish among homozygous wild types homozygous mutants and heterozygous individuals based either on the C or RFU value RFU data may be chosen from any cycle in the experiment The assignments can be made automatically if controls are specified or you can make the assignments manually NOTE The Allelic Discrimination module is not available with working with single color data files or any data file generated by the MyiQ Real Time PCR Detection System 6 7 1 Allelic Discrimination Plot The Allelic Discrimination plot shows either RFU Figure 6 28A or C Figure 6 28B data from two different fluorophores at the same time Choose which plot to view using the RFU or Threshold Cycle radio button in the Display Mode box In Automatic Call mode two bars one vertical and one horizontal divide the plot into four sections one for each homozygous state one for the heterozygo
207. tions in the PCR Quant tab in a number of ways In the upper corner of each section is a plus button that enlarges the section when clicked The enlarged section has a minus button that reduces the section when clicked You can move divider bars between each section by clicking and dragging on the divider bar to resize panes to a specific size 6 2 Amplification Chart The amplification chart Figure 6 2 displays the relative fluorescence for each well at every cycle Each trace represents the fluorescence of a given fluorophore for a single well and at each cycle a single data point is plotted which is the calculated mean of the data collected for that well during the particular cycle The data that is used to determine this mean point is set by the Set Data Analysis window dialog box The data can be plotted in Background Subtracted PCR Base Line Subtracted or PCR Base Line Subtracted Curve Fit mode Amplification Chart Fig 6 2 The Amplification Chart 6 2 1 Fluorophore Selector Buttons Selecting Fluorophores to Display You can use the fluorophore selector buttons which are located under the amplification chart to display which fluorophores appear in the amplification and standard curve charts Selecting a single fluorophore is useful for determining the analysis parameters for that fluorophore Selecting all fluorophores can be useful for ensuring that the efficiencies of each fluorophore s
208. tions to the instruments not performed by Bio Rad or an authorized agent Table of Contents NOTICE TO PURCHASER eres enne renes randa ad ainakaan iAH inanasan Ra uuu a ada i Safety Information eerie eiie serene rennen anna nnne n nanus a annu auam u aug R aaa amu au una uuu ai u annus ii Section 1 Getting Started t cee 1 1 1 The MyiQ 2 Real Time PCR Detection System eeseseeeeeenenenenne nnnm 1 1 2 Setting Up the System Hardware esee nnnm enne nnn nnn n nnn nnn nnn nnn 1 us System Checklist fc e R 1 1 2 2 Installing the Optical Reaction Module on the iCycler Chassis eeeese 2 1 2 3 Installing the Support Bracket eeeeeeeeeneneneneene nennen nnn nn nnne 3 1 2 4 Installing the Optics Module seessessssssesese eene ener nennen nennen 3 1 2 5 Connecting Power and Communication Cables to the System uuususss 3 1 3 Installing the iQ5 Software 0 0 i i i i i i a i en na a a a a a nnn nter rne nnne 4 1 3 1 Installing the Camera Drivers sse 4 1 4 Recommended Computer Settings sssssssssseseeeee eene 4 1 5 Calibrating the Instrument seini eire tette tank e nee ann an peek eaa aaa a a akku a aea 6 1 6 Compatibility with Earlier Versions of the iQ5 Optical System Software
209. tions to this problem e Use Bio Rad iQ SYBR Green supermix which contains fluorescein enabling dynamic well factors to be collected directly from the experimental plate e Use persistent well factors e Spike reaction mixes that do not come premixed with fluorescein with fluorescein to a final concentration of 10 nM The addition of fluorescein provides sufficient fluorescence at 95 C for the collection of well factors from the experimental plate while not interfering with the PCR reaction 5 3 1 Spiking Real Time PCR Experiments Using DNA binding Dyes With Fluorescein The iQ SYBR Green supermix is already spiked with a small amount of fluorescein that permits the collection of well factors from the experimental plate It is also possible to collect well factors from the experimental plate with other commercial SYBR Green mixes or with home brew mixes by adding sufficient fluorescein to bring the reaction mixture to 10 nM fluorescein Prepare a 1 mM solution of fluorescein by making a 1 1000 dilution of the 1 mM stock fluorescein calibration dye in PCR buffer 10 mM Tris pH 8 0 50 mM KCI 3 mM MgCl Then mix 1 part of the 1 mM fluorescein with 990 ul of master mix to yield a final concentration of 10 nM fluorescein Once well factors are collected from the experimental plate they are written to the opd file and the software continues to execute the programmed protocol 5 4 Initiate Run Window Run End Point Selected Initiate a
210. tocol editing options by selecting Run time Protocol Editing as shown below Run time Protocol Editing Selecting this checkbox activates the Next Cycle and Add 10 Repeats buttons as shown below Next Cycle Add 10 Repeats e Next Cycle Click Next Cycle to complete the current repeat of the present cycle before skipping to the next cycle For example you could use this feature when your samples have clearly crossed threshold and you want to skip to the melt cycle of your protocol 45 Section 5 Run Time Central Module Add 10 Repeats Click Add 10 Repeats to add additional repeats to the current cycle You can click this button multiple times however the total number of repeats is limited to 600 For example it may be necessary to add repeats to a run in an experiment amplifying low copies of DNA to allow all samples to cross threshold Click Add 10 Repeats to an amplification cycle of 30 repeats to make it 40 repeats Modifications to the protocol are updated on the protocol displayed on the iCycler base module Pause Stop Resume Run The Pause Stop Run allows you to pause a thermal cycling protocol If you click Pause Stop Run when the iCycler thermal cycler is at a setpoint temperature the instrument will hold at the setpoint temperature and stop counting down the dwell time If you click Pause Stop Run when the iCycler cycler is ramping to the temperature the instrument will continue ramping until it reaches the next
211. trator roles The Administrator can customize the roles of a Principal Operator and Guest by checking or unchecking the box associated with each function and role To apply the selected changes click Save Role Changes To restore factory default roles click Restore Factory Roles NOTE In the factory default roles users assigned to the iQ5 Guest role do not have any permission granted to them By default these users are granted read only access to the software unless the ability to start runs and save files is granted by the iQ5 Administrator Viewing User Permissions To view the current permission settings for existing iQ5 Users access the User Administration window by clicking on the Administration button All non administrator users will have read only access to the information in the User Administration window 132 Section 9 Instrument Maintenance Section 9 Instrument Maintenance This section contains information on the following topics o Cleaning the system page 133 o Recommended fluorophores and filter specifications page 133 o Accessing and cleaning the filters page 134 o Replacing the lamp page 135 9 1 Cleaning the Real Time PCR Detection System Take care not to spill liquids onto or into the iCycler thermal cycler or the optical module Cleaning may be done using a lint free cloth or paper towel The case may be cleaned using a soft lint free cloth and water 9 2 Filter Description and Installa
212. ts of the following three choices Negatives Positives and Positives amp Negatives Analysis by comparison to negative controls Negatives is the default method Ranks The number of ranks allows assignment of samples into distinct groups based on their RFU values The default Rank value is 10 and the minimum number of Ranks is 3 e End Cycles to Avg End Cycles to Average is the number of cycles from the last cycle that will be used to calculate an average end point RFU value The End Cycles to Avg field defaults to 5 for non end point only PCR runs e End Point Only Run The End Cycles to Avg field defaults to 2 for end point only runs e Tolerance Mode End Point Tolerance defines the margins for sorting unknowns as positives or negatives The End Point Tolerance drop down list box consists of two choices RFUs and Percent of Range e Tolerance 9 o Select a value between 1 and 99 percent for this setting The default percent of range tolerance is 10 percent e Color Scheme Colored rank boxes symbolize the number and order of ranks in the end point analysis There are five options to choose from that allow a change in the color scheme of the rank boxes once the data are analyzed 127 Section 8 User Profiles 8 1 8 Gene Expression Module Preferences The Gene Expression module preference box Figure 8 9 allows you to predetermine several analysis and display options associated with analyzing C values for gene expres
213. u can see the progress of the run in this window At the end of the run the Run Status dialog box appears You can choose between displaying the data in the Data Analysis module or returning to the Workshop module Click Yes to proceed to the Data Analysis module 2 4 Data Analysis Quick Guides When the iQ5 software opens the Data Analysis module is grayed out and inactive To analyze a data file click the Data File tab of the Workshop module select the data file and then click Analyze The Data Analysis module consists of six tabs e PCR Quant e Melt Curve Peak e End Point e Allelic Disc e Gene Expr e Edit Plate 2 4 1 PCR Quant Tab Quick Guide Use the PCR Quant tab to set the analysis conditions for the data file including setting the PCR baseline setting the threshold and determining which wells to exclude or include in the experiment To analyze a data file 1 Click the Data File tab in the Workshop module 10 Section 2 Quick Guides Select a data file from the file tree browser and then click Analyze The file opens in the PCR Quant tab within the Data Analysis module Select or deselect wells to be included in the analysis by selecting Analyze Wells Select or deselect wells to be displayed by selecting Display Wells The iQ5 software automatically chooses the data analysis conditions including baselines and thresholds If the data file is being opened for the first time an automated analysis of base
214. udecea bedi EXR aaia ga aaiae 131 8 3 2 Changing PassWord sssini aisanana tpe a RARE VXRRRRERRR NALE a aAA Aaa AAAA AAE a aaia aaRS 131 8 3 3 Defining ROIES uniin repehner a a ERTa SR ERR ELA FREE CERES 132 Section 9 Instrument Maintenance enses esee ener n 133 9 1 Cleaning the Real Time PCR Detection System sss enne 133 9 2 Filter Description and Installation Instructions eeeeeeeeeeeeeeennn mmm 133 9 2 1 Recommended Fluorophores and Filter Specifications eeeeeeeeeeeeees 133 9 2 2 Accessing the A eS 2 pr Rt Le ERR ERR EREEE ARR eR aea aaia 134 9 2 3 Cleaning the Filters deret aiiai Teo kenn cececeveeducaendnddeeteancedactiecedcens 134 9 3 Replacing the Lamp z odo corni nec Ere e E 135 Appendix A Warranty cccccsssccseseesssseeeeseeeeseeenaseeneseeaeasesneaeeaeaeeeesseeneaeeseaseesseesseasensenee Appendix B Troubleshooting Error Messages eeeeneeeee ener nnn Appendix C Product Ordering Information eene eeeen nnne nnne nnne viii Section 1 Getting Started Section 1 Getting Started This section contains information on the following topics o The MyiQ2 Real Time Detection System page 1 o Setting up the system hardware page 1 o Installing the iQ5 software page 4 o Recommended computer settings page 4 o Compatibility with earlier versions of the software page 6 1 1 The MyiQ
215. ugh the recorded fluorescence of each well during the baseline cycles The iQ5 software then subtracts the best fit data from the background subtracted data at each cycle to generate the PCR baseline subtracted trace By default the software automatically chooses the beginning and end baseline cycles You can override this default and manually give each trace a beginning and ending baseline cycle User specified settings for PCR baseline subtraction may also be specified in the User Preferences module PCR Base Line Subtracted Curve Fit The iQ5 software fits the PCR baseline subtracted data to a smoothed curve using a balanced flank centroid finding digital filter The curve fit process is performed in such a way that the C is left invariant for all traces 6 2 3 Log View Button You can click Log View shown below to change between a semi logarithmic and linear display of the amplification chart data BLog View 50 Section 6 Data Analysis Module 6 2 4 Selecting and Viewing Traces Identifying a specific amplification trace Identify a specific trace by moving the mouse pointer along the trace until the hand icon appears The dialog box identifies the trace by both well name and fluorophore in the top left corner of the amplification chart Selecting a specific amplification trace Select a specific trace by moving the mouse pointer on the trace until the hand icon appears and then double click The dialog box displays the selec
216. urn the camera on and restart the iQ5 software Check that the USB cable is securely connected to both the camera and computer Ensure that the 2 0 high speed enabled USB port on the computer is being used and all hibernate and power save settings are disabled Refer to section 1 2 5 and 1 4 for further details I x A Failed to start run The plate setup that you have chosen current seal Film vessel Plates base unit Io2Emulator and camera IQ2ColorEmulator does not match any of the currently stored Background Factors calibration Files Please review the current calibrations under View gt Calibration files gt BackgroundFactors and recalibrate your instrument BB Show error details Cause Background factor data not found This error occurs when a run start is attempted using a vessel and seal combination the instrument has not been calibrated for Solution The background vessel and seal type selected for your plate setup must match collected background calibration files The instrument should ideally be re calibrated for the desired vessel and seal type combination If a run must be started immediately select a vessel and seal type for which the instrument has already been calibrated After the run has completed recalibrate the instrument for the desired vessel and seal combination and back apply this new background calibration to your data file Refer to section 4 5 5 for further details 138 Appendices x
217. us state and a non reactive section The positions of these bars may be adjusted in the Automatic Call mode The bars do not appear in Manual Call mode 69 A Section 6 Data Analysis Module Allele1 B Allele2 a Heterozygote None Controli Control2 Allele1 B Allele2 a Heterozygote None Controli m Control2 RFU for Allele 2 HEX 6000 55 40004 gt m Threshold Cycle for Allele 2 HEX 2000 4 a T 1 TIT 1000 1000 2000 3000 4000 25 30 35 40 45 50 55 RFU for Allele 1 FAM Threshold Cycle for Allele 1 FAM Fig 6 28 Allelic Discrimination Plot RFU View A and Threshold Cycle View b Genotype assignments for unknown samples are determined by plotting the RFU for one fluorophore allele 1 on the x axis against the RFU for the other fluorophore allele 2 on the y axis on the allelic discrimination plot If the unknown RFU values are greater than the horizontal and vertical bars then the genotype is heterozygous If the unknown RFU values are greater than the horizontal bar and less than the vertical bar the genotype is homozygous for allele 2 allele 2 RFU is plotted on the y axis If the unknown RFU values are less than the horizontal bar and greater than the vertical bar the genotype is homozygous for allele 1 allele 1 RFU is plotted on the x axis If the unknown RFU values are less than the horizontal and vertical bar
218. ve Peak module of the iQ5 software including the melt curve chart melt peak chart Melt spreadsheet and analysis parameters End Point Reports e End Point detailed When creating a report from a data set that includes PCR Quantification data End Point detailed is the only report option available This report includes a section titled Data Analysis Parameters which details the PCR analysis settings for the current dataset End Point only When creating a report from a data set performed as an End Point Only run the End Point Only report is the only report option available This report template contains only the End Point Analysis spreadsheet and data analysis parameters Allelic Disc Reports Allelic Data Only The Allelic Data Only report template is limited to the data displayed in the Allelic Disc module only but will not display the allelic discrimination chart Allelic Detailed This report template includes all of the data available in the Allelic Disc module of the iQ5 software including the allelic discrimination chart spreadsheet and analysis parameters In addition if all fluorophores have been selected for display in the PCR Quant module the PCR amplification chart and analysis parameters will also be displayed Allelic Only This report template is limited to the data displayed in the Allelic Disc module only and includes the allelic discrimination chart spreadsheet and analysis parameters Gene E
219. ven gene If multiple genes have an identical range of dC values then 3 Setthe dominant calibrator to the gene with the smallest absolute value of Average dC of eligible inter run calibrator samples If multiple genes have identical Average dC absolute values then 4 Set the dominant calibrator to the replicate group with the smallest dC NOTE The first data file imported into the Gene Study will always serve as the hub file for pair wise data comparisons during Inter run Calibration F Inter run calibration Select gene F Inter run 1 HEX 2 HEX calibrator 118 2092 18 2156 25 7935 25 3787 22 5125 22 5923 29 5600 29 7665 33 4224 33 3596 12 6251 12 5211 15 1694 15 5875 Average dct ddct 4 b bl 1 HEX vs 2 HEX 1 HEX vs 3 HEX OK Fig 6 61 The Inter Run Calibrator Calculation Display 104 Section 6 Data Analysis Module Details of the Inter run Calibration calculations can be accessed by clicking the Inter run Calibration button The resulting Inter run Calibration window Figure 6 61 will display the comparative fluorophore calculations per gene pair wise comparisons e You can choose to view the inter run calibrator calculations for different genes in your assay by selecting a Gene Name from the Select Gene drop down menu e You can choose to view the calculations resulting from different pair wise comparisons by clicking on the desired tab displayed below the spreadsheet Inter
220. xpression Reports e Gene Expression Detailed This report template includes all the Gene Expression data available in the Gene Expr module including charts data tables Gene List and Condition List information Threshold Crossing Spreadsheet and Gene Expression analysis 109 Section 6 Data Analysis Module parameters In addition if all fluorophores have been selected for display in the PCR Quant module the PCR Amplification Chart and PCR Baseline Analysis Parameters will also be displayed Gene Expression Graph only This report template displays only the Gene Expression chart exactly as analyzed and formatted within the gene expression module Gene Expression only This report template includes all of the data tables Gene List and Condition List information Threshold Crossing Spreadsheet and Gene Expression analysis parameters available in the gene expression module The gene expression chart is NOT displayed Gene Study Report MultiFiles Gene Expression Detailed When creating a report from within a Gene Study MultiFiles Gene Expression Detailed is the only selection available This report format is similar to the Gene Expression Detailed report however no PCR Quant data is available for this report 110 Section 7 Calibrating the Instrument Section 7 Calibrating the Instrument This section contains information on the following topics o Calibration overview page 111 o Components required for calib
221. ycles are indicated with a blue background To insert a step click in the Insert column on a Step row Steps are indicated with a white background e Deleting Cycles and Steps To delete a cycle click in the Delete column on the Cycle row Cycles are indicated with a blue background To delete a step click in the Delete column on a Step row Steps are indicated with a white background e Saving the Protocol To save a protocol click Save amp Exit Protocol Editing Enter the name of the protocol in the Save As dialog box and click Save again 2325 Section 4 Workshop Module The Protocol Editor may only be exited by clicking Save amp Exit Protocol Editing or Cancel amp Exit Protocol Editing 4 3 3 Add Protocol Options If you want to add any protocol options first enable them by clicking in the check box next to its description in the Show Options box The Protocol Options include e Gradient see section 4 3 4 for details e Infinite Hold e Ramping e Temperature Change e Time Change e Cycle Description e Step Process Infinite Hold When a cycle is not repeated you can specify the dwell time at any step in that cycle as infinite by using the Infinite Hold option This means that the instrument will maintain the specified temperature until execution is interrupted When an infinite dwell time is programmed within a protocol at some step other than the last step the instrument will go into Pause mode when it reaches that
222. ymbols It can be of any length An Administrator can also change the password of users This is useful if a user forgets the password To Add a new user 1 Type the desired login name into an empty User Name box of the Defining Users spreadsheet Optional Type the full name of the user Define the Role of the user by using the pull down menu in the Role cell Select Administrator Operator User or Guest The features functions that each of these user roles is permitted restricted to use is defined by an Administrator using the Defining Roles spreadsheet Optional Type the email or phone contact information of the user Optional Type the password for the user This password defined here by the Administrator can later be changed by the user using the Change Password option found in the Tools menu Click Save User Changes 129 Section 8 User Profiles Adding a user will create a folder with the new user s name This folder will be created in the Bio Rad iQ5 Users folder and will be the default folder for all files saved when the user is logged into the software 8 2 2 Deleting Existing Users Users are removed using the Defining Users spreadsheet 1 In the Defining Users spreadsheet check the box in the Delete column for the user that is to be removed 2 Click Save User Changes NOTE No messages will appear to confirm this action The User Profile will immediately be removed from the Defining Users spread

Instruction Manual, iQ5 Optical System Software - Bio-Rad

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