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qPCRsoft Software for REAL-TIME PCR Thermal

1. m Ag E Empty Group 1 ao B Empty Group 1 aun Empty Group 1 1 l F Table Standard curve Fig 49 Window for absolute quantification e Parameter settings e Display of fluorescence spectra e Display of sample table with analysis results e Display of the regression lines and the calculated coefficients 58 07 2012 qPCRsoft Evaluation 5 2 2 Parameter settings for absolute quantification AbsQuant Autom Threshold qPCRsoft Abs Quantification AbsQuant Gene of interest GOT FAM c myc Group Threshald P Indude pass reference Group 1 3 923 Fig 50 Parameter settings for absolute quantification Set the following parameters for the absolute quantification Option SELECTION LIST GENE OF INTEREST GOI INCLOUD PASS REFERENCE GROUP THRESHOLD Description Selection of an evaluation created for the experiment Selection list of target gene dye combinations According to the selection the fluorescence and regression curves for the concentration are displayed Only active if a dye has been defined as a passive reference on the SETTINGS SCAN project tab If this option is activated the fluorescence of the dye that has been set as a passive reference is used for standardization If several experiments were carried out on the PCR plate select the group of the experiment to be analyzed gt section Defining groups p 41 Manually adjust thresho
2. ccccececeececeeeeeeeeeaeeeseeeeseees 85 5 6 3 Specifying genotyping Options cece ceecceeeeeeeeseeeeseeeeaeeeeeeeees 86 5 6 4 Displaying the fluorescence curves scatter plot and bar graph 87 5 6 5 Display of the values for the genotyping evaluation 0 89 5 6 6 Deleting a genotyping cccccceecceecceeeceeecceeeceeeceeeceeeseeeseeeseeeses 91 Functions in EXTRAS MENU cccccccescseeceeeecceeeeeseesensensonseneeeeseeeees 92 6 1 Device initialization a5 coche ce luctea eli coe tla kssweobind ani ehs soca siiucuaatedaiiats 92 6 2 Opening the lid TOWEROnIY 0 ccc cceccceeeeeeeeeeeeeeseeeeaeeesaeees 92 6 3 Exchanging the block QF OWERONIY ccceccseececeeeeeeeeeeeeeeaes 92 6 4 Editing Color MOUIES cccccccecceecceeecceeeeeeeceeeceeecseecseeseeeseeeses 92 6 5 Connecting the device to the PC 1 00 eccccccc cece eeeeeeeeeeeeaeeesneeaees 93 07 2012 qPCRsoft qPCRsoft Contents 6 6 General settings in the gPCRsoft software cccccceseeeeeeeeeees 93 Working with the user management 0 ccsseseeeeeeeeeeeeeeeeeeees 95 7 1 LISGE QlOUDS iccncticodas dates a aa a a a 95 7 2 Managing USer PrOmleSys cs icdsaceieeass n a eas vadendunl seeds 96 7 2 1 Adding a ser profile ss cces ercecs eile ltlate eds a reese 96 7 2 2 Editing a user profile Changing a password cceceeseeeeeeeees 97 7 2 3 Deleting a user profile
3. cccceccsecseeeeeeeeees 36 Entering sample properties into the sample table 37 Displaying sample properties in the project explorer 0 38 Entering a sample layout for a multiplex assay ccceeeeeeees 38 Entering a sample layout for a singleplex assay 000 000n0000000n 40 3 4 2 HSTINING Or OUDS iaa e ele eso Redes 41 3 4 3 Layo OFC VICW indicat itd avant doiatinl Macitenduaeauea dita candi dawtielalaadundeiiautedalmes 43 3 4 4 CODVIING TNS NAY OU aine aiuanltenatelqtecientebdeneetateasented ame tetess 43 3 4 5 Exporting or importing the layout in Excel c cccceeeseeeeeeeeeeees 44 07 2012 1 Contents NIOHILOMING iia oie inet ontesvenboswotatieied a 46 4 1 Starting the PCR protocol ccccccccesecceeeeceeeceeeeseeeseeesseeseeeens 46 4 2 Display Options for MONILOLING cccccecceseeeeeeeeceeeeaeeeseeeeeeeeseues 47 4 2 1 Default settings for the Monitoring VICW cccccccseeeeeeeeeeeeeseees 48 4 2 2 Adjusting the view in the MONITORING project WiIndowW 0 48 4 2 3 Displaying and hiding measurement results for individual wells 49 4 2 4 Exporting fluorescence data cccccccccceeceeeeeeeeeecaeeeseeeeseeeeaeeeeaes 50 4 3 Monitoring the PCR run a oannannnannnnnnennsnnnnnnnnrnsnennrnnnernnrsnrrerernrrnnee 51 4 4 Displaying product accumulation curves ccecceeccseeeaeeeneeeneeeees 52 4 5 Display
4. 3 4 1 Settings Samples project tab 36 Defining sample properties Entering sample properties in the layout You can define the properties for the samples in the wells on the SETTINGS SAMPLES project tab in the layout view and the edit area next to it Editlayout Create groups H Unit ng Fig 25 Layout view for the sample type The following sample types can be defined Sample type SymbolDefinition EMPTY Describes an empty position on the PCR plate UNKNOWN T Sample of unknown concentration or dilution measuring sample STANDARD B Sample of known concentration or dilution CALIBRATOR K Sample the target gene expression level of which is set to 1 Complete reaction mixture but without template NO TEMPLATE CONTROL N i strand NTC POSITIVE CONTROL Positive control assay for which a reaction product can be expected NEGATIVE CONTROL Negative control assay for which no reaction product is to be expected Samples with identical sample properties Sample name sample type same gene dye assignments are viewed as multiple measurements The individual values of these samples are averaged and their mean value is used for the remaining calculations With a singleplex assay samples can have the same sample name and sample type but differ as far as the gene dye assignment is concerned These samples are identified as associated samples due to the same name The evaluation however
5. cccecccecccceeecceeeceececeeeeceneeceeeeeeeseueessueess 97 VNC EEE EE EEA E EE E E A E A E E E 98 07 2012 3 Contents 4 07 2012 qPCRsoft 1 1 1 1 2 qPCRsoft qPCRsoft Software qPCRsoft Software The qPCRsoft software can be used to create and perform PCR and Real Time PCR experiments This chapter describes the basic setup and the layout of the operating elements of the software Described software version This description is based on the version qPCRsoft V 1 0 Intended use The qPCRsoft software serves for controlling the qT OWER and evaluating the data recorded with this device The manufacturer does not assume any liability for problems or damage caused by the unintended use of qPCRsoft qPCRsoft and the device to be controlled by it may only be operated by appropriately qualified and instructed personnel The user must be familiar with the information given in this manual and in the user manual of the hardware How to use this manual The following symbols and conventions are used to facilitate orientation in the manual Cross reference to other sections and or figures Formatting In the description of the operating procedures menu commands dialog boxes buttons options etc are highlighted in small capitals Menu commands of a command sequence are separated by slashes e g File Open project Buttons are additionally written in square brackets e g SAVE Installation of qPCRsoft
6. Administrator rights on the operating system are required for installing the program System requirements for installing qPCRsoft For using qPCRsoft to control the Real Time PCR device your PC must fulfill the following minimum requirements Operating system Windows XP SPS2 VISTA 7 Processor Pentium IV gt 1 GHz RAM 1 GB Available hard disk space min 300 MB Interfaces USB 07 2012 5 qPCRsoft Software Installation procedure qPCRsoft is delivered on CD Rom 1 Insert the CD in the CD Rom drive Normally the installation s start window opens automatically If this is not the case start the setup exe file on the CD A selection dialog window for installing the device driver or the user management or for viewing the PDF files of the manuals appears 2 Activate your language version Click on INSTALL The installation routine begins 4 Follow the further instructions of the installation program 5 Switch on the device at the power switch Start qPCRsoft Note The software will only be installed correctly if it has been started once with administrator rights A password for the program administrator must be entered during this first start 1 3 Starting and exiting qPCRsoft File Exit Starting qPCRsoft 1 To start qPCRsoft click on the START button on the Windows desktop Then go to the PROGRAMS folder and search for QPCRSOFT Click on QPCRSOFT there 2 Alternatively you may click on the qPCRsoft
7. Depending on the selected window size and the number of dyes to be measured two buttons with left and right arrows appear They can be used to move between the different tabs The number of columns that are displayed in the sample table can be user defined Right click on a column header to display the following selection field v Well Well color Name Type color Type Comment 7474 44 ks Group name Fig 28 Selection field for defining the columns displayed in the sample table You can select or deselect individual columns to show or hide a column and modify the representation as desired The order of the columns can be changed by clicking on the table header and dragging with the mouse Displaying sample properties in the project explorer Moving the mouse pointer over a well on the SAMPLE project tab in the layout view will display the properties of the well in the SAMPLES menu item in the project explorer J Samples Position Group Name Type Genel Dyel Fig 29 Project explorer Samples window showing the well properties Entering a sample layout for a multiplex assay The following example shows the definition of four samples and standards with three repeat measurements each in the layout The GAPDH gene is analyzed with the FAM dye and the c myc gene with the VIC dye The two dyes are selected on the SETTINGS SCAN project tab and activated for measurement The indicated sample names and standa
8. 4 2 4 Exporting fluorescence data The data from the fluorescence measurement can be exported as csv files In addition the graphical display of the measurement results can be copied to the clipboard as a hardcopy and is hereby made available for other programs Q Right click on the graph Q A selection window for export and hardcopy appears Q Click on COPY CHART to copy the chart to the clipboard E Select the SAVE CHART option to export the fluorescence data The SAVE AS standard window opens Enter a file name and confirm with OK 50 07 2012 qPCRsoft 4 3 qPCRsoft Monitoring Bi Realtime per project 3Toptical Demo _AbsQuantl_L rtp f fm 6 Settings Monitoring 2 Analysis db Measurement tid 25 0 _4 steps scan a pf 95 0__ 02 00_ Table Graph T Profile Fig 39 Context menu for exporting the fluorescence data in the MONITORING project window Monitoring the PCR run The running PCR protocol is shown in the bottom part of the MONITORING project tab It is generally possible to to choose between the three different views list sheets GRAPH TABLE and T PROFILE via tabs In addition to the display on the list sheets a status bar shows further information on the protocol such as the plateau time the calculated remaining time and in programmed loops the step number and the number of loops Schritt 3 von 3 Loop 11 von 40 Ge Plateauzeit 8 s Restzei
9. Button Command Function General NEw Opens a new project ry al OPEN TEMPLATE Opens a template Lt SAVE TEMPLATE Saves a template a g OPEN PROJECT Opens a project Lt SAVE PROJECT Saves a project g PRINT PROJECT Prints a project 3 UNDO Reverses the last modification a REDO Restores the last deleted modification d CUT Cuts a marked area 10 07 2012 qPCRsoft qPCRsoft COPY PASTE xX DELETE PCR protocol ADD EMPTY STEP DELETE STEP ACTIVATE MELTING CURVE mT CUT STEP TA i COPY STEP S PASTE STEP Color Ha EDIT COLOR COMPENSATION Samples EDIT LAYOUT COPY LAYOUT Ill Pi PASTE LAYOUT PREVIEW LAYOUT Monitoring gt START PCR 7 PROTOCOL L STOP PCR PROTOCOL i OPTIONS T qPCRsoft Software Copies an active and or marked area Pastes an area copied into the clipboard Deletes an active and or marked area Adds a new step Deletes a step Adds a step for melting curve determination Cuts a step and copies it to the clipboard Copies the parameters in one step into the clipboard Pastes a copied step Opens the window for creating files for spectral color compensation Assigns changes that were made to the sample table Copies an area in the sample table Pastes a copied area from the sample table Displays a complete view of the plate assignment Starts the PCR run Ends the PCR run Display options of the product accumulation curves Evaluation absolute
10. Comment Fig 5 Real time PCR run General information Command Description TITLE Analysis title OPERATOR User If you are using a user management option the user name you signed in with will be entered automatically DAY TIME Date the project was created COMMENT Comment input Note Edit For text inputs on the tab GENERAL being able to use the usual commands for qPCRsoft copying cut out and insert of texts These commands are arranged in the menu EDIT 07 2012 19 Settings for a Real Time PCR experiment 3 2 Creating aPCR protocol Settings Thermocycler project window 20 A PCR protocol must be programmed for each Real Time PCR experiment All necessary functions are combined in the SETTINGS THERMOCYCLER project window The screen is divided in three sections graphical program preview 1 program header 2 and program table 3 Real time per project 3Toptical_Derno_AbsQuantl_1 rtp lo lt iS Settings 2 Monitoring EM Analysis 4 p T General fi Thermal Cycler Scan im Samples db 0 1000 2000 3000 Time s Fam able T Step 1 of 4 2 cater c 100 2 Wirebesth deve Fop r i sises e e gets ees Te fats 20s Table Graph Melting curve Fig 6 Program table for creating PCR protocols The program preview illustrates the history of the PCR protocol The program header defines the general conditions for the PCR protocol such as the programmed lid t
11. H4 H6 Std 4 c myc 10 15 Click on ma to assign the sample properties to the three values Note Connected samples must have the same sample name v The plate layout for a singleplex assay is complete 3 4 2 Defining groups qPCRsoft Several experiments can be performed on one microplate at the same time The samples that are part of one experiment are combined in a group A group contains a number of reaction mixtures that will be evaluated together later on You can define a maximum of 12 such groups The groups are defined in the SETTINGS SAMPLES project window 1 In the SETTINGS SAMPLES project window click on the CREATE GROUPS button 07 2012 41 Settings for a Real Time PCR experiment The GROUP list and the GROUP NAME field are activated In the layout all samples are marked with the number 1 This means they have been assigned to group 1 2 Inthe layout mark the samples that are part of one experiment To mark adjacent samples press and hold the mouse button Select the next group from the GROUP list 4 Enter the description for the experiment in the GROUP NAME field You may select any group name 5 Click on a to assign the group properties to the samples The samples that belong together are marked with the group number in the layout The descriptions are displayed in the sample table in the GROUP NAME column 7 Real time per project ject Unnamed o ED Settings Monitoring LB Anal
12. SCAN project tab The standard setting for the color compensation is OFF This is due to the fact that color compensation is not required for the most common applications only one active measuring channel or dyes that are spectrally widely spaced such as FAM and ROX If you click on SELECT a window opens up in which color compensations that have already been recorded can be opened and used again In the window only those compensation data are printed in black color which meet the settings on the card scan Invalid compensation data appear in red color and cannot be selected 32 07 2012 qPCRsoft Scan Edit color compensation qPCRsoft Settings for a Real Time PCR experiment E Real time per project tst rtp TF Settings Wa Monitoring Ed Analysis w E 1 Blue 470nmi S2O0nmi SybrGreen 5 2 Green 515mm 545mm JOE 5 gt 3 Yellow 535mm sanm TAMRA 5 gt 4 Orange 56Snm 605m Texashed 5 lt gt 5 Red 630nm 670n0m cys 5 6 MIRi 660mm 7O5nm vod 5 Meas repeats 3 Color compensation Select f Scan region ac Ce Reagan A EE X Define scan rec Cc itamratr CCB3_ 1bis4 Fig 22 Window for selection of color compensation data Select a color compensation for the current project with the left mouse button and press OK to activate that compensation With the symbol or the menu command SCAN EDIT COLOR COMPENSATION you can create a new color compensation by measurement This process is called spectral c
13. THRESHOLD menu command Whether you choose manual or automatic calculation the resulting threshold value is updated and displayed synchronously in the corresponding THRESHOLD input field 5 2 3 Displaying the fluorescence curves for absolute quantification 60 In the display range the measured data standardized to the value 100 for highest fluorescence intensity is plotted against the cycle for the selected target gene The respective fluorescence curves are displayed by switching to another target gene dye combination The fluorescence data is displayed as a linear or logarithmic representation depending on the selected display option For both display options the program shows brief information on the sample if the cursor is placed on one of the curves Switching the display options for the chart 1 Click on the 2 button in the parameter bar A selection window for the display options opens 2 Select the SCALING LOGARITHMIC or LINEAR option Click next to the selection window The changes are applied Zyklus Fig 51 Linear representation of the fluorescence curve for the absolute quantification 07 2012 qPCRsoft Evaluation Zyklus Fig 52 Logarithmic representation of the fluorescence curve with horizontal threshold line 5 2 4 Displaying the standard curve and results of an absolute quantification In the bottom STANDARD CURVE section of the ANALYSIS project window you can switch between the calculated s
14. The device initialization sets the device to the original state A device initialization is only required after an error has occurred Extras Device LJ Call up the EXTRAS DEVICE INITIALIZATION command initialization 6 2 Opening the lid qrOWERonly The opening and closing functions of the qf OWER are motorized Extras Open LJ Call up the EXTRAS OPEN CLOSE LID command close lid 6 3 Exchanging the block qTOWERonly To exchange the block the thermoblock must be moved up This movement must be software controlled Extras Change LJ Call up the EXTRAS CHANGE BLOCK command block 6 4 Editing color modules After inserting the color modules into the device s probe the color modules must be specified in the software Edit color modules x olor modules 7 Eigenschaften Blue 470 520 11 3 Position Green 515 545 11 2 ji Yelow 535 580 11 2 E Orange 565 605 11 2 Blue 470 520 11 3 Red 630 670 11 2 MIR 1 660 700 11 1 Add Delete Accept Standardmadule zur cksetzen Fig 88 Edit color modules window Extras Edit color 1 Call up the EXTRAS EDIT COLOR MODULES command modules A window with the same name opens 2 The available color modules for your device are displayed on the left 92 07 2012 qPCRsoft Functions in Extras menu From the list select the module you have installed in the device activate the PROPERTIES checkbox and select the position on which the modu
15. but also any other cycle of the PCR run The respective cycle can be selected from a list NAME Input fields for own names for the wild type mutant heterozygote E categories or otherwise SCATTER Generation of the scatter plot based on the fluorescence intensities PLOT of the analyzed cycle and or the Ct values 5 6 4 Displaying the fluorescence curves scatter plot and bar graph The respective combination of wild type dye or mutant dye displayed is shown on the tabs in the bottom left corner of the area The entry for the respective active combination on the tab is highlighted in white The fluorescence data is displayed as a linear or logarithmic representation depending on the selected display option For both view types a brief information is shown as soon as the mouse pointer is positioned on one of the curves 1 Click on the button in the parameter bar A selection window for the display options opens 2 Select the SCALING LOGARITHMIC or LINEAR option Click next to the selection window The changes are applied qPCRsoft 07 2012 87 Evaluation 100 T3 Fig 83 Linear representation of the fluorescence curve for genotyping Wildtyp FAM Mutante ROX Scatter Plot End Point Fig 84 Logarithmic representation of the fluorescence curve with horizontal threshold line To determine Ct values for the analysis a threshold value must be determined for the fluorescence curves This can be automate
16. depending on the end point fluorescence or Ct value Assign replicates according to wild type mutant heterozygote or error symbol Standardized fluorescence intensity of the wild type reaction Standardized fluorescence intensity between replicates of the wild type reaction Standard deviation of the standardized fluorescence intensity between replicates of the wild type reaction Standardized fluorescence intensity of the mutant reaction 07 2012 qPCRsoft Evaluation MEAN DRN MUTANT Standardized fluorescence intensity between replicates of the mutant reaction STD DEV DRN Standard deviation of the standardized fluorescence intensity MUTANT between replicates of the mutant reaction Individual columns can be shown or hidden by selection or deselection The layout of the columns can also be freely modified By dragging a column heading with the left mouse button depressed columns can be swapped The display of the results in the table can thus be adjusted as desired 5 6 6 Deleting a genotyping Genotyping Delete Genotyping qPCRsoft An evaluation that is no longer required can be removed 1 Activate the evaluation by selecting its name in the evaluation list of the method tab Click on re in the toolbar Alternatively call up the GENOTYPING DELETE GENOTYPING menu command The evaluation is removed 07 2012 91 Functions in Extras menu 6 Functions in EXTRAS MENU 6 1 Device initialization
17. 07 2012 2 Settings for a Real Time PCR experiment LJ Another option for adjusting the gradient is to click on the top end of column 6 and change the height of the column with the left mouse button depressed Moving the column the changes the annealing temperature accordingly Gradient Step 1 of 4 Annealing temp aC Increment AIE oC oc 64 3 64 5 65 1 66 0 67 0 68 0 69 0 70 0 71 0 71 9 72 5 72 7 rwl1 213 4tsle6l71s19 iu Table Graph Gradient Melting curve Fig 17 Programming a linear gradient 3 2 4 Graphical display and programming the PCR protocols Settings In the SETTINGS THERMOCYCLER project window the history of a programmed PCR Thermocycler protocol is displayed graphically in the TABLE and GRAPH screens It represents the Graph project temperature curve of the block blue line and the heating lid red line over time window The green diamond symbolizes steps during which the fluorescence is measured Temp C 500 1000 1500 000 2500 Zeit s Fig 18 Graphical representation of a PCR protocol Programs are generally created in the table view which allows new steps to be programmed quickly and provides a summarized overall survey of the protocol structure Some programming options are only available in the table view The graphical programming mode additionally offers a schematic representation of the temperature profile and the option to adjust pro
18. 1 33 89 33 8 D Empty Group 1 Empty Group 1 Unknown Group 1 32 9 33 2 0 Fig 79 Genotyping window e Parameter setting for defining the threshold and selecting the dye with which the wild type and the mutant were measured 84 07 2012 qPCRsoft Evaluation e Display of the fluorescence curves and or display of the results as a scatter plot or bar graph e Display of the sample table with the results 5 6 2 Parameter settings for genotyping Group Threshold Indude passive reference Group 1 30 539 o Fig 80 Parameter settings for genotyping for the fluorescence curve display Set the following parameters for the absolute quantification Option Description SELECTION LIST Selection of an evaluation created for the experiment WILD TYPE Selection list for the dye with which the wild type was measured MUTANT Selection list for the dye with which the mutant was measured INCLUDE PASSIVE Only active if a dye has been defined as a passive reference REFERENCE on the SETTINGS SCAN project tab If this option is activated the fluorescence of the dye that has been set as a passive reference is used for standardization GROUP lf several experiments were carried out on the PCR plate select the group of the experiment to be analyzed gt Defining groups page 41 THRESHOLD Manually adjust threshold value The threshold value must be between 1 and 100 depending on the standardized representation of the fluoresc
19. 2 To save files enter the name of the template in the standard window and save the template with OK Templates are saved with the extension rts File Save The changes in a template can be saved with the FILE SAVE TEMPLATE menu template l l Lt command Optionally you can click on the SEE symbol in the toolbar 2 3 Saving a project You can save the project with all parameters of the PCR run the fluorescence curves and evaluations File Save project 1 Call up the FILE SAVE PROJECT AS command oo 2 To save files enter the name of the template in the standard window and save the template with OK Projects are saved with the extension rtp File Save project The changes in a project can be saved with the FILE SAVE PROJECT menu command Optionally you can click on the be symbol in the toolbar 2 4 Importing exporting analyses Settings for the data evaluations of a project can be saved exported and later transferred imported to an open project The evaluations are applied to the open project when they are imported 16 07 2012 qPCRsoft Managing projects File Export 1 Select the FILE EXPORT ANALYSES menu command analyses 2 To save files enter the name of the analysis and save the files with OK Analyses are saved with the extension rta File Import 1 Select the FILE IMPORT ANALYSES menu command aliases 2 Select the name of the analysis in the default window for opening
20. 54 basic settings 54 Export fluorescence data 57 Genotyping see Genotyping Melting curve see Melting Curve 07 2012 Relative quantification see Relative quantification sample selection 56 AACt method see AACt method Extras menu Change block 94 Device identification 95 Device initialization 94 Edit color modules 95 Open close lid 94 Options 96 F File menu Close 17 Close all 17 Exit 6 Export analysis 17 Import analysis 17 New 15 Open autom saved project 16 Open project 15 Open template 15 Print 17 Save project 17 Save project as 16 Save template 16 Save template as 16 Fluorescence measurement measurement parameters 30 PCR protocol 25 G Gene 37 Genotyping create evaluation 86 delete evaluation 93 end point bar graph 91 results 91 Scatter plot 90 set parameters 87 special evaluation options 88 threshold values 88 view fluorescence curve 90 Genotyping menu Add genotyping 86 Autom threshold 88 Delete Genotyping 93 Options genotyping 55 88 Groups 41 Installation qPCRsoft procedure 6 system requirements 5 qPCRsoft qPCRsoft L Language settings 96 Layout copy 43 Excel import export 44 preview 1 42 groups 41 view 34 Lid temperature 21 Livak method 75 Melting curve create evaluation 80 delete evaluation 85 display 53 display melting curves 82 Melting temperature 84 program 26 set parameters 81 threshold value 82 Melting curve menu Add melting curve 8
21. S Enter the increment or decrement of the hold time within the PCR run A C S Enter the heating or cooling rate to reach the target temperature in the temperature step Inserting a new temperature step deleting a temperature step Cycler Add empty LY You can insert an additional temperature step to the program table with the WV step arrow key Optionally you can add another temperature step with the CYCLER ADD EMPTY STEP menu command oro The total number of steps and the current processed step are displayed in the protocol header Cycler Delete pd step LI To delete a program step move the cursor to the program row and click on Optionally you can call up the CYCLER DELETE STEP menu command Entering the target temperature hold time and heating cooling rates LI Enter the target temperature of the temperature step into the C column Q Into the m s column enter the hold time in the format minutes seconds e g hold time of a duration of 1 min 20 s 1 20 qPCRsoft 07 2012 23 Settings for a Real Time PCR experiment LJ For special applications or the transfer of protocols from other devices it may be required to adjust the heating and cooling rates Enter the average heating and cooling rate for each individual step in the A C s column Note The value in the A C s column defines the speed at which the target temperature is reached If the temperature is be heated or cooled at a speed of 3
22. assigned to each well in the sample table SETTINGS SAMPLE project window 07 2012 qPCRsoft Monitoring The measurement results for the individual dyes are selected via the corresponding list sheets You have the option to display the measurement results of all dyes together ALL COLORS list sheet or only the results of an individual dye The display options for the product accumulation curves are described in the Display options for monitoring section on page 47 4 5 Displaying melting curves To display the melting curves select the MELTING CURVE option from the VIEW list The display options for the product accumulation curves are described in the Display options for monitoring section on page 47 qPCRsoft 07 2012 53 Evaluation 5 Evaluation On the ANALYSIS tab of the project window the following methods are available for evaluating Real Time PCR experiments on four cards e Absolute quantification e Relative quantification e AACt method e Melting curve determination e Genotyping Ej Real time pcr project demo_qiOWER rtp z ioj x Settings Monitoring Analysis Abs Quant Rel Quant DeltaDeltact Melk curve Genotyping Abs Quantification AbsQuant Gene of interest GOT Group Threshold FAM c myc Include pass reference Group 1 3 923 R 2 0 9987 Slope 7 96 Offset 93 39 Efficiency of PCR 7 48 2 L5 l 0 5 0 0 5 1 Log Concentration Standard c
23. choose manual or automatic calculation the resulting threshold value is updated and displayed synchronously in the corresponding THRESHOLDinput field 5 4 3 Displaying the fluorescence curves for the AACt method qPCRsoft In the display range the measured data standardized to the value 100 for highest fluorescence intensity is plotted against the cycle for the selected target gene The gene dye and the reference gene dye combinations are each assigned a list sheet that can be activated by clicking on the gene dye tab on the bottom In the evaluation you can always use only one target gene but several reference genes The number of the available list sheets depends on the number of the selected genes The fluorescence data is displayed as a linear or logarithmic representation depending on the selected display option For both display options the program shows brief information on the sample if the cursor is placed on one of the curves Switching the display options for the chart 1 Click on the 2 button in the parameter bar A selection window for the display options opens 2 Select the SCALING LOGARITHMIC or LINEAR option Click next to the selection window The changes are applied dRn GOI AM Ref genFAM Fig 65 Linear representation of the fluorescence curve for the AACt method 07 2012 73 Evaluation GOI AM Ref gen FAM Ref gen FAM Fig 66 Logarithmic representation of the fluorescence curve for the
24. files and import the analysis into the current project by clicking OK 2 5 Closing project windows File Close The FILE CLOSE menu command closes the active project window To close all project windows select the FILE CLOSE ALL menu command If any unsaved File Close all changes have been made in project windows a confirmation prompt appears 2 6 Printing You can specify the desired contents for printing a project in a selection list File Print 1 Select the FILE PRINT menu command 2 Manage the print output using the displayed lists Select the desired information in the list on the left and click Can transfer it to the print list on the right To remove undesired information from the print list click 3 Click PRINT to start the printout Select OPTIONS to configure the printout or PREVIEW to display a page view of the print image tE Amplification 2 E Melting curve fO Analysis E Abs Quant Eg Rel Quant lit DeltaDeltaCt 7 Melt curve Fig 4 PRINT window The individual print modules are sorted into the SETTINGS MONITORING and ANALYSIS subgroups in the PRINT window qPCRsoft 07 2012 17 Managing projects 2 7 Signing projects Edit Signatures 18 Projects can be signed if the user management has been activated 1 Activate the project you would like to sign 2 Select the EDIT SIGNATURES menu command to open the SIGNATURE window 3 Click LOGIN and log
25. for the display options opens 2 Select the SCALING LOGARITHMIC or LINEAR option Click next to the selection window The changes are applied 07 2012 qPCRsoft Evaluation j Well E2 25 40 18 Leber Zyklus i GOI FAM Fig 58 Linear representation of the fluorescence curve for the relative quantification Zyklus Fig 59 Logarithmic representation of the fluorescence curve for the relative quantification with horizontal threshold line 5 3 4 Displaying the standard curves and the results of a relative quantification In the upper area of the project window you can switch between the display of the calculated standard curves for the target gene and all selected reference genes and the sample table for the relative quantification using the STANDARD CURVE and TABLE list sheets For the display of the standard curve the Ct values of the standard samples are plotted graphically against the logarithm of their concentration In the value range on the right the following calculated data is displayed e the coefficients of determination R of the linear equation e the standard curve gradients e the intersections of the curves with the y axis at x 0 offset e The PCR efficiency The standard curve and the values are automatically calculated by the qPCRsoft software and updated in case of settings modifications According to the number of genes used a scroll bar appears under the table It can be used to navigate through the t
26. icon on the Windows desktop qPCRsoft is started If the user management has been installed you will be prompted to enter user name and password The qPCRsoft workspace will only become accessible if the entry of this data was successful Note An administrator and password must be defined during the first program start Only the administrator can set up further user accounts or disable the user management Exiting qPCRsoft 1 To exit the qPCRsoft application activate THE FILE EXIT menu command 2 The program will display a message if any projects that have not yet been saved are still open at this point 3 If you want to save these projects click on YES Save the projects in the SAVE AS standard window 4 Then call up the FILE EXIT menu command again to exit qPCRsoft 07 2012 qPCRsoft qPCRsoft Software 1 4 The main window in qPCRsoft GS qPCRsoft 1 1 Edit View Extras Window Help 1 PSD DDDB S E boo a UUN E Thermal se 3 Scan EJ Samples Rel Quant i DeltaDetact B i Mett Curve Curve E Fig 1 Main window of the qPCRsoft software After starting the qPCRsoft software the main window opens It has the following sections Menu bar 1 The menu bar contains the menu commands for e g opening editing and saving projects managing user profiles setting basic software options and a help function Toolbar 2 Commands for editing projects are arranged in the toolbar T
27. in with your USER NAME and PASSWORD 4 Select a status from the list DOCUMENT SEEN DOCUMENT APPROVED DOCUMENT IN PROGRESS or DOCUMENT REJECTED 5 Confirm with OK 07 2012 qPCRsoft Settings for a Real Time PCR experiment 3 Settings for a Real Time PCR experiment If you want to start a new project create a new project or open a template LJ Create an empty project with in the toolbar LJ Optionally open a template with Tto use the previously stored parameters for the new project or to modify them All functions necessary to create a new project are combined under the SETTINGS tab Additional tabs on the second level are assigned to the SETTINGS tab Tab page Function GENERAL Allows you to enter general information and remarks THERMOCYCLER Used for programming PCR protocols SCAN Definition of the colors to be measured and settings for the measuring parameters SAMPLES Opens the sample table in which detailed information on each sample can be saved and groups of experiments defined 3 1 Entering general information on the project Settings General You can save general information on each project The entries can be made on the project window GENERAL tab Real time per project 3Toptical Dermo_AbsQuantl_1 rtp Lo EY Settings Monitoring EM Analysis d gt EL General i Thermal Cycler Scan Samples q p Tite Demo data set for absolute quantitation Operator Winter Day Time 11 07 2012 Ir 00 00 00
28. is performed individually Q Mark the sample position to be edited by clicking into the layout Multiple selections are only possible in adjoining fields To do so press and hold the left mouse button and move the cursor over the corresponding fields To mark rows or columns click on the corresponding row or column name A H or 1 12 You can mark all sample positions in the layout by clicking on the gray button in the top left of the layout between 1 and A1 07 2012 qPCRsoft qPCRsoft Settings for a Real Time PCR experiment Fig 26 Button for marking the complete layout LI Enter the following sample parameters in the adjacent edit area Parameter Description SAMPLE NAME Sample description SAMPLE TYPE Selection of the sample type see top of table TARGET table E i GENE column In the dye row enter the gene to be analyzed CONC column For standards Enter the concentration of the gene to be analyzed LI You can assign sample properties to the marked positions by clicking on the symbol in the toolbar Note The entries for the selected area will only be applied by the program after they have been assigned Entries or modifications that are not assigned will be lost LI To display defined sample properties in the edit area double click on a well You can edit the information and assign them to the well again by clicking ma To assign these sample properties to other wells m
29. properties to the three wells Mark the three wells A4 A6 Make the following settings Parameter Entered value Sample name Sample 1 Sample type Unknown FAM c myc Click on Ea to assign the sample properties to the three wells Repeat steps 3 8 for the other three samples Use the following parameters Wells Sample name Sample type Gene FAM B1 B3 Sample 2 Unknown GAPDH B4 B6 Sample 2 Unknown c myc C1 C3 Sample 3 Unknown GAPDH C4 C6 Sample 3 Unknown c myc D1 D3 Sample 4 Unknown GAPDH 07 2012 qPCRsoft Settings for a Real Time PCR experiment D4 D6 Sample 4 Unknown c myc Defining the standard samples 10 Mark the three wells E1 E3 11 Make the following settings Parameter Entered value Sample name Std1 Sample type Standard Make the following entries in the Target table Dye Gene Conc FAM GAPDH 100 Note The CONC column in the TARGET table is only available for the STANDARD sample type 12 From the UNIT list choose a concentration or mass unit You can select from the following units ng ng ul ng ml pg ul copies copies ul copies ml mg ml U ul U ml or 13 Click on Ea to assign the sample properties to the three wells 14 Repeat steps 10 13 for the other standards Use the following parameters Wells Sample name Gene Conc E4 E6 Std 1 c myc 100 F1 F3 Std 2 GAPDH 75 F4 F6 Std 2 c myc 75 G1 G3 Std 3 GAPDH 50 G4 G6 Std 3 c myc 50 H1 H3 Std 4 GAPDH 10
30. standard window opens Enter a file name and confirm with OK Real time pcr project 3Toptical_Demo_AbsQuantl_1 rtp o em ES Settings a Monitoring ES Analysis 4 b Ee Abs Quant a Rel Quant Wt DeltaDeltact IE Melt curve db Pie ee ee a Uanttaton 1 Gene of interest GOI Group Threshold Indude pass reference Group 1 1 156 GOI FAM Fig 48 Exporting data from the evaluation qPCRsoft 07 2012 57 Evaluation 5 2 Absolute quantification The absolute quantification is used to determine absolute copy numbers in samples with the help of the comparison with standards with known concentrations 5 2 1 Creating an evaluation for an absolute quantification AbsQuant Add 1 Goto the ANALYSIS ABS QUANT project tab abs quantification If the tab is not visible click on the arrows 4 t in the tab bar This will scroll the tabs 2 Click on the 7 symbol in the toolbar Optionally call up the ABSQUANT ADD ABS QUANTIFICATION menu command 3 An input window appears Enter the description for the current evaluation On the ABSQUANT tab the following information is activated Real time pcr project 3Toptical_ Demo_AbsQuantl_1 rtp oc a _ fis Abs Quant g Rel Quant DeltaDeltact Melt curve 4b Group Threshold Indude pass reference Group 1 1 156 k
31. the following areas Real time per project 3Toptical_Derno_AbsQuant1_1 rt per p ject pt P iy Settings Monitoring G Analysis db P 50 wo 950 00 05 _ 580 00 05 _ 72 0 00 15 4 able Graph T Profile Fig 35 Monitoring project window for monitoring PCR runs Area Function MEASURING Displays the measured fluorescence data The fluorescence RESULTS 1 intensity is plotted against the cycle COLORS tabs 2 Switches between the fluorescence accumulation curves that were measured for the individual dyes PCR PROTOCOL Displays the PCR protocol The active step is indicated by a 3 green arrow PROTOCOL VIEW Switches between different views of the PCR protocol tabs 4 tabular graphic temperature profile The fluorescence measurements are displayed in the top area On the different list sheets you can choose between the overlapping display of the measurement results of all dyes or the display of the individual dyes In the VIEW list you can switch between the AMPLIFICATION MELTING CURVE and RAW DATA views The current PCR protocol is displayed at the bottom of the table in the project window The active step is marked by means of a green arrow during the PCR run On the different sheets you can choose between a tabular or graphical view of the PCR protocol or the temperature profile The view of the PCR protocol is described in the section Monitoring the PCR run
32. window for basic settings for the evaluation Automatically determines the threshold Different menus 2 in the project explorer offer a quick overview of the currently processed project Individual projects can be selected via a selection list 1 The information on the individual menus can be displayed or hidden via the and 3 buttons 07 2012 qPCRsoft View Project explorer qPCRsoft Software O Samples i ZJ 4 5 amp F amp F W Gi i i 2 r te E E E Fig 2 Organization of the project explorer Menu Information GENERAL Project title user date timeand device THERMOCYCLER Graphic display of the history of the PCR program in the active project SCAN Overview of the colors and areas of the PCR plate that are being scanned SAMPLES Displays a short info text on the plate layout The edit mode for the plate layout displays detailed information on the selected well ABSOLUTE Graphic display Ct against log concentration QUANTIFICATION RELATIVE Graphic display Ct against log concentration QUANTIFICATION AACT MELTCURVE GENOTYPING Graphical display dCt V compared to log concentration Graphical display of melting curve compared to temperature Graphical display of dRn wild type compared to dRn mutant as a scatter plot or bar graph You can display or hide the project explorer via the VIEW PROJECT EXPLORER menu command 1 4 4 Project interface with project window qPCRsoft The
33. 0 Autom threshold 82 Delete melting curve 85 Options melting curve 55 Menu commands 7 Monitoring 46 default settings 47 Export fluorescence data 50 monitore PCR run 51 sample selection 49 view 46 Monitoring menu Display options 48 Pause qPCR run 46 Start qPCR run 46 Stop qPCR run 46 View options 46 Multiplex assay 38 P PCR experiment comments 19 generell informations 19 PCR protocol create 20 delete step 23 edit 22 fluorescence measurement 25 grafical programming 28 heating cooling rates 23 hold time 23 insert new step 23 melting curve 26 menu commands 21 program header 21 program loop 24 target temperature 23 07 2012 Index temperature and time decrement 24 temperature and time increment 24 Temperature gradient 27 PCR run start 46 Pfaffl method 75 Product accumulation 52 Project automatically save 96 from template 15 new 15 opening 15 opening automatically saved project 16 printing 17 saving 16 signing 18 viewing 16 Project explorer Abs Quant 63 Meltcurve 85 overview 12 Rel Quant 70 sample parameters 38 Samples 49 Samples 56 AACt 78 Project window 13 Q qPCRsoft exiting 6 starting 6 R Relative quantification create evaluation 65 delete evaluation 70 display fluorescence curves 67 results 69 set parameters 66 standard curves 68 threshold value 67 RelQuant menu Add rel quantification 65 Autom Threshold 67 Delete rel quantification 71 Import standard curve 70 Options rel qu
34. 1 Manually defining the scan region Project window Settings Scan qPCRsoft The scan region can be defined according to the plate layout in the sample table see section Editing the sample table p 34 or manually The scan region for the thermocycler is always defined per column It must always consist of connected columns 1 Fora manual sample selection select the DEFINE SCAN REGION MANUALLY option on the SETTINGS SCANtab 2 A graphical representation of the sample block is opened Enter the first and last column of the region to be scanned into the FROM COLUMN andTO COLUMN fields 07 2012 31 Settings for a Real Time PCR experiment Optionally you can use the mouse to select the columns To select an individual column click directly into that column If you wish to select several columns press and hold the left mouse button and move the cursor over the corresponding area Active columns are highlighted blue in the diagram E Scan region according layout Define scan region manually a on oat Fig 21 Manual adjustment of the scan region 3 3 2 Color compensation Project window If you are using several dyes per reaction mixture the result may be a fluorescence Settings Scan crosstalk This means that a second dye is excited and measured next to the desired dye at the same time To subtract the fluorescence quotient of the second dye you can use the color compensation function on THE SETTINGS
35. AACt method 5 4 4 Setting the calculation mode for the standardized expression 74 The qPCRsoft software enables you to calculate the standardized expression SE with two different methods e Without PCR efficiency calculation Livak method e With PCR efficiency calculation for GOI and reference genes Pfaffl method To calculate the standardized expression SE one sample must be defined as the calibrator Calculating the standardized expression SE without efficiency calculation Livak method The Livak method assumes that the PCR efficiencies of the gene of interest GOI and the reference gene RefG are equal The following applies NE 2 AACt where AACt ACt Calibrator ACt Sample and ACt Sample Ct GOI Sample Ct RefGene Sample ACt Calibrator Ct GOI Calibrator Ct RefGene Calibrator Calculating the standardized expression SE with efficiency calculation Pfaffl method The Pfaffl method considers the efficiencies determined for the gene of interest GOI and the reference gene RefGene The efficiencies E GOI and E RefGene can be calculated from dilution series or specified in the software The following applies E GOI SoG NE E Ref Gene S Refeene where ACt GOI Ct GOI Calibrator Ct GOI Sample 07 2012 qPCRsoft 9 4 5 qPCRsoft Evaluation and ACt RefGene Ct RefGene Calibrator Ct RefGene Sample The Pfaffl method is generally preferred as the
36. C per second between step 2 and step 3 the value 3 0 must be entered for step 3 Note If the speed is to be reduced for the whole program the heating or cooling rates of all steps must be adjusted The settings are only valid for that one program 4 steps scan oC mis goto loops ATEC Atis Zec LC 95 0 05 00 oe He ME hea Ee aE Ee L an oma a ee CORNE Fig 9 Entering the target temperature hold time and heating cooling rates in the PCR table Defining loops Program sequences that are repeated regularly can be summarized in loops Generally a loop is then defined by a target step for the return GOTO and the number or repetitions LOOPS Tan onan 2 29 so O T T SN T T Fig 10 Programming a loop in the PCR table 1 Place the cursor on the last step of the future loop step 4 in the example above 2 Enter the number of the target step into the GOTO column 2 in the example above 3 Inthe LOOPS column enter the number of repetitions 39 in the example above After entering the target step and the repetitions the programmed loop is displayed as a bracket on the left side of the table see figure above Note The total number of loops displayed in the bracket is determined from the number of programmed repetitions plus 1 as the corresponding sequence of steps prior to reaching the loop has already cycled once Entering increments decrements for temperature and hold tim
37. FAM GAPDH Position fellkultur 24h Zellkultur 24h ruppe 5 FAM G amp POH FAM GOPOH Mame FTellkulkur 24h fellkultur 74h felikultur 24h Typ Standard g FAM MCF 1 FAM MCF 1 Zellkultur 74h ea 24h Gent GAPDH Konz i00ng U 1 FAM MCF 1 FAM MCF 1 FarbstorFi FAM Fig 31 Section from the layout preview table The table can be printed and used for example as a template for pipetting the samples or for documenting the experiment LJ Press the PRINT button in the VIEW SAMPLE PLATE window to print the table 3 4 4 Copying the layout Samples Copy layout Samples Paste layout qPCRsoft The layout view or parts of the layout can be copied and inserted at different positions in the same layout or into the layout of another project 1 Use your mouse to mark the area in the layout view to be copied Transfer the information to the clipboard by clicking on the Pe symbol in the toolbar Optionally call up the SAMPLES COPY LAYOUT menu command 3 Select a position for inserting the copied area The selected position or the left upper position of the selected area determines the position at which the copied section in the layout view is to be inserted Paste the information using the JE symbol Optionally you can use the SAMPLES PASTE LAYOUT menu command 07 2012 43 Settings for a Real Time PCR experiment The way described to edit the layout is related to the graphical presentation of the PCR
38. IVE QUANTIFICATION The image displays the graphical plot of the Ct values of the standard samples against the logarithm of their concentration F Rel Quant Fig 62 Representation of the standard curve in the project explorer 5 3 5 Importing the standard curve for relative quantification Next to the option to measure a standard curve in the experiment the qPCRsoft software can also be used to determine the concentration of the samples based on the saved standard curve You can use the import function for this purpose 1 Usethe at symbol in the toolbar to open the IMPORT STANDARD CURVE window RelQuant Import Optionally you can call up the RELQUANT IMPORT STANDARD CURVE menu standard curve command 2 All additional setting are analogous to the settings for the absolute quantification see section Importing the standard curve S 63 qPCRsoft 07 2012 69 Evaluation 5 3 6 Deleting the evaluation of a relative quantification An evaluation that is no longer required can be removed RelQuant Delete 1 Activate the evaluation by selecting its description in the evaluation list of the rel quantification method tab es 2 Click onthe symbol in the toolbar Alternatively call up the RELQUANT DELETE REL QUANTIFICATION menu command The evaluation is removed 70 07 2012 qPCRsoft Evaluation 5 4 AACt method The AACt method allows for determination of the relative expression level of a gene of i
39. ME Name of sample SAMPLE TYPE Type of sample GROUP Assignment of the sample to an experimental group GENE Name of gene measured in the sample CT Ct value of sample MEAN CT Mean Ct value of replicates CONC STD Concentration of the standard sample MEAN CONC Concentration determined from the standard curve on the basis of the mean Ct value STDDEV CT Standard deviation of the Ct values between replicates YCV CT Variation coefficient of the Ct values between replicates The individual columns can be shown or hidden by selecting or deselecting them Moreover the columns can be rearranged freely To exchange columns press and hold the left mouse button and drag a column header to the desired location The display of the results in the table can thus be customized individually Display in the project explorer A shortened representation of the standard curve calculated by the software is displayed in the project explorer under ABS QUANT The image displays the graphical plot of the Ct values of the standard samples against the logarithm of their concentration i 15 2 25 3 35 4 Fig 55 Representation of the standard curve in the project explorer 07 2012 qPCRsoft 9 2 9 Importing the standard curve Evaluation Next to the option to measure a standard curve in the experiment the qPCRsoft software can also be used to determine the concentration of the samples based on the saved standard curve You can use the import function f
40. SCADE HELP Menu CYCLER SCAN SAMPLES MONITORING ABSQUANT RELQUANT qPCRsoft CONTENT INFO fashion qPCRsoft Software Arranges project windows in a cascaded Opens the table of contents of the help function Displays software information Function ADD EMPTY STEP DELETE STEP CUT STEP COPY STEP PASTE STEP EDIT COLOR COMPENSATION EDIT LAYOUT COPY LAYOUT PASTE LAYOUT PREVIEW LAYOUT START QPCR RUN STOP QPCR RUN PAUSE QPCR RUN DISPLAY OPTIONS ADD ABS QUANTIFICATION DELTE ABS QUANTIFICATION OPTIONSABS QUANTIFICATIONG AUTOMA THRESHOLD IMPORT STANDARD CURVE ADD REL QUANTIFICATION DELTE REL QUANTIFICATION OPTIONSREL QUANTIFICATIONG AUTOMA THRESHOLD IMPORT STANDARD CURVE 07 2012 Description Adds a new step Deletes a step Cuts a step and copies it to the clipboard Copies the parameters in one step into the clipboard Inserts a copied step Opens the window for creating files for spectral color compensation Edit sample table Copy the area of the sample table Insert the copied area of the sample table Detailed view of the plate assignment Start the PCR run Stop the PCR run Pause the PCR run Display options for the product accumulation curves Create new evaluation Delete evaluation Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold for detecting Ct values Import a s
41. able s columns qPCRsoft 07 2012 67 Evaluation FAM GAl R2 0 99943 a Slope 3 7 20 Offset 32 5 15 PCR Efficiency 0 9 0 36 1 2 3 4 Log Std Conc 4 lo p Table Standard curve Fig 60 Standard curves for relative quantification The sample table for the relative quantification contains all data and the associated measurement values for the samples B6 Empty Group 1 B7 fee ti Standard Group 1 26 53 26 46 ct BB std Standard Group 1 26 5 26 46 ct Bg o Std 1 Standard Group 1 26 35 26 46 ct Bio P Empty Group 1 si B Empty Group 1 i 68 Table Standard curve Fig 61 Sample table for relative quantification The selection of the displayed columns can be user defined Right click on a column header to display the following selection field Column WELL COLOR OF CURVE SAMPLE NAME Meaning Position of sample Each sample is automatically assigned an unchangeable color which is used to display the corresponding fluorescence curve Name of sample SAMPLE TYPE Type of sample GROUP Assignment of the sample to an experimental group GOl Gene of interest REFERENCE GENE CT GOl CT REFERENCE GENE Reference gene Ct value of gene of interest Ct value of reference gene MEAN CT GOI Mean Ct value of replicates of the gene of interest MEAN CT REF GEN Mean Ct value of replicates of the reference gene CONC STD GOI Concentration of the standard for the gene of interest CONC STD Concentration of the standard fo
42. agement 7 2 Managing user profiles User profiles can only be set up by a user with administrator rights Users may change their password if allowed by the administrator 7 2 1 Adding a user profile Edit User 1 Click on the EDIT USER MANAGEMENT menu command to open the USER management PROFILES window 2 Click ADD Enter the following data in the USER PROFILE window Option Description GENERAL tab USER NAME Login name at program start FULL NAME Real name optional DESCRIPTION Further details optional USER GROUP Assign user group with the respective rights PASSWORD tab 96 07 2012 qPCRsoft PASSWORD CONFIRM PASSWORD USER MUST CHANGE PASSWORD WITH NEW LOGIN USER MAY CHANGE PASSWORD No PASSWORD TIME OUT USER IS DEACTIVATED USER IS LOCKED Working with the user management Enter password Repeat password If activated the user must change his password after the first login The administrator allows the user to change his own password Password is valid for an unlimited time If deactivated the expiry date must be specified User profile has been locked by the administrator or because the wrong password was entered three times User profile has been locked by an administrator 7 2 2 Editing a user profile changing a password 1 Click on the EDIT USER MANAGEMENT menu command to open the USER PROFILES window 2 Mark the user pro
43. alibration A new window opens in which all the required settings can be made The window is divided in a selection list for dyesand a plate diagram Color Compensation i x Color module oye 1 ahahah ah ah Mame MyFAM_JOE_HEX_Calibration m Fig 23 Color compensation for spectral calibration window To record the calibration data the dyes required for a color compensation must be available individually in solution You could for example use the sensors for the 07 2012 33 Settings for a Real Time PCR experiment calibration measurement that are to be used in the later PCR experiment The dye concentration for the calibration measurement should be approx 0 1 umol l In the displayed plate diagram the wells that contain the calibration samples are now marked individually for each dye and the dye in each of the samples is assigned to the marked well by clicking on the blue arrow The dyes offered for selection are those that were selected in the SETTINGS SCAN project tab For an exact calibration measurement we recommend that each dye be created at least as a triple replicate as well as at least three blanks sample containers filled with water The calibration measurement is started by clicking on the blue triangle on the bottom left gt Note The selection of available dyes cannot be modified in this table Modifications can only be made on SCAN tab The new color compensation needs to be assigned a descripti
44. alysis The program shows brief information on the sample if the cursor is placed on one of the curves The melting temperature Tm is determined by forming the first derivative of the melting curves from the maxima of the forming peaks ddRn dT 60 65 70 fo 80 85 90 Temp C Derivative GOI AM Fig 75 View of the melting curves derivative with threshold line qPCRsoft 07 2012 81 Evaluation You can switch between the melting curves display and the derivatives via the tab in the bottom left corner of the display range Switching the display options for the chart of fluorescence curves 1 Click on the button in the parameter bar A selection window for the display options opens 2 Select the SCALING LOGARITHMIC or LINEAROoption Click next to the selection window The changes are applied The DERIVATIVE tab cannot be displayed logarithmically dRn 60 65 70 5 80 85 90 95 Temp C Derivative Gor FAM Fig 76 Fluorescence curves display linear view The linear display does not show a threshold line 5 5 4 Displaying the sample table for the melting curves The sample table for melting curves contains all data and the associated measurement values for the samples B6 B smp2 Unknown Group 1 77 4 T Tabe Fig 77 Sample table for melting curve analysis The selection of the displayed columns can be user defined Right click on a column header to display a selection f
45. ame Sample name Sample type Sample type Group Group Ct Wild type Genotyp Mean Ct Wild type Reaction Wild type Std Dev Ct Wild type vw Reaction Mutant Ct Mutant Genotyp Replicates Mean Ct Mutant v dRn Wild type Std Dev Ct Mutant Mean dRn Wild type Std Dev dRn Wild type dRn Mutant Mean dRn Mutant Std Dev dRn Mutant Fig 87 Context menu for displaying the values in the sample table for genotyping Column Meaning WELL Position of sample COLOR OF CURVE SAMPLE NAME SAMPLE TYPE GROUP CT WILD TYPE MEAN CT WILD TYPE STD DEV CT WILD TYPE CT MUTANT MEAN CT MUTANT STD DEV CT MUTANT GENOTYPE REACTION WILD TYPE REACTION MUTANT GENOTYPE REPLICATES DRN WILD TYPE MEAN DRN WILD TYPE STD DEV DRN WILD TYPE DRN MUTANT Each sample is automatically assigned an unchangeable color which is used to display the corresponding fluorescence curve Name of sample Type of sample Assignment of the sample to an experimental group Ct value of wild type Mean Ct value of replicates of the wild type Standard deviation of the Ct values between replicates of the wild type Ct value of mutant Mean Ct value of replicates of the mutant Standard deviation of the Ct values between replicates of the mutant Assign the sample according to wild type mutant heterozygote or error Yes or no depending on the end point fluorescence or Ct value Yes or no
46. analytikjena aPCRsoft Software for REAL TIME PCR Thermal Cycler Operating manual analytikjena Service Analytik Jena AG Customer Services U Konrad Zuse Str 1 TH RINGEN 07745 Jena r CERT _ ISO 9001 2008 15 100 6153 Germany Phone Hotline 49 0 3641 77 7407 Fax 49 0 3641 77 7449 Email service analytik jena de General information about Analytik Jena AG on the internet http www analytik jena de Copyrights and Trademarks multi N C and multiWin are registered trademarks of Analytik Jena AG in Germany Microsoft Windows XP VISTA 7 MS Excel are registered trademarks of Microsoft Corp The identification with or TM is omitted in this manual Edition July 2012 Implementation of the Technical Documentation Analytik Jena AG This publication describes the state of this product at the time of publishing It need not necessarily agree with future versions of the product Modifications reserved Copyright 2012 Analytik Jena AG qPCRsoft Contents Contents 1 aPCRSOft SOM AN Caisse eiaraheciicieneecac aaaea aaa aai pA AAE A 5 1 1 How to use this MANUAL wisn cccecevvacsiawisee decd clita oodseevaaieawienieeeide 5 1 2 Installation Ol GP CIRSOMN siririna ieia E 5 1 3 Starting and exiting QGPCRSOft cc ecccceecceeeeeeeeeseeeeseeeeaeeesaeeees 6 1 4 The main window in QPCRSOft ccccccccseceeseeeseeeeseeeeseeeeseeesaeeees 7 1 4 1 Menu commands overview c ccc
47. antification 55 S Sample parameters 35 project explorer 38 Sample table edit 34 Sample types 35 Samples menu Copy layout 43 Paste layout 43 99 Index 100 Preview layout 42 Scan menu Edit color compensation 33 Scatter plot 90 Signatures 18 Singleplex assay 40 Standard concentrations 37 T Temperature program see PCR protocol Template creating 16 Thermoblock exchange 94 Threshold automatic 55 Tool buttons 10 U User management activating deactivating 96 User groups 97 user profiles manangement 98 V View menu Project explorer 13 Toolbar 10 A AACt method create evluation 72 delete evluation 79 display fluorescence curves 74 results 77 set parameters 73 standard curves 77 standardized expression 75 threshold value 73 validation curves 76 07 2012 qPCRsoft
48. ard curve Fig 56 Window for relative quantification 07 2012 qPCRsoft Evaluation e Parameter settings e Display of the fluorescence curves for the target gene and the reference gene e Display of the standard curves for the target gene and the reference gene and the calculated coefficients e Display of the sample table with the added values 5 3 2 Parameter settings for relative quantification qPCRsoft Rel Quantification Gene of Interest GOT FANM c myc ER Group Threshold Include passive reference Group i 23 163 Reference genes VIC GAPDH 3 Fig 57 Parameter settings for relative quantification The following parameters must be set for the relative quantification Option SELECTION LIST GENE OF INTEREST GOI REFERENCE GENES INCLUDE PASS REFERENCE GROUP THRESHOLD Description Selection of an evaluation created for the experiment Selection list of target gene dyecombinations According to the selection the fluorescence and regression curves for the concentration are displayed Only one target gene at a time can be selected Reference gene selection list In contrast to the target gene several reference genes can be selected at the same time The number of list sheets in the display range therefore grows with each reference gene The symbol is used to remove all reference gene settings from the evaluation Only active if a dye has been defined
49. ark the desired wells and then click E The entry of a sample layout is described by means of an example for a singleplex assay Entering a sample layout for a singleplex assay S 40 and a multiplex assay Entering a sample layout for a multiplex assay S 38 Entering sample properties into the sample table You can also make entries in the sample table itself Select the desired position in the layout view or a field directly in the sample table The corresponding row is then highlighted yellow Descriptions and or values can be defined directly in the designated cells The sample table is edited cell by cell Multiple selections and the associated assignment of parameters to several cells or rows at a time are not possible Standardkonze Group 1 ae zellkultur 24 Standard Group 1 E zellkultur 24 Standard Group 1 Zz zellkultur 24H Standard Group 1 E zellkultur 24 Standard Group 1 E Unbekannt Group 1 E Unbekannt Group 1 Unbekannt Group 1 Fig 27 Sample table for entering sample properties 07 2012 37 Settings for a Real Time PCR experiment The genes and in the case of standard samples their concentration are summarized separately by dye in the second part of the sample table A list sheet is assigned to each dye It is thus possible to use different standard concentrations for each gene The number of displayed sheets depends on which dyes have been activated on the SETTINGS SCAN project tab for this measurement
50. as a passive reference on the SETTINGS SCAN project tab If this option is activated the fluorescence of the dye that has been set as a passive reference is used for standardization If several experiments were carried out on the PCR plate select the group of the experiment to be analyzed gt Section Defining groups p 41 Manually adjust threshold values The threshold value must be between 1 and 100 depending on the standardized representation of the fluorescence curves dRn Note The threshold value can be calculated automatically or set in the graph Opens the selection window with display options Setting the threshold value To determine Ct values for the evaluation a threshold value needs to be determined for each experiment first You have several options for setting the threshold value 07 2012 65 Evaluation RelQuant Autom Threshold e Inthe general options see section Making basic settings p 54 e Manually in the parameters for the respective evaluation see table above e Graphically in the fluorescence curves representation e By having it calculated automatically LJ Inthe chart move the black threshold line up or down with the cursor Press and hold the left mouse button while doing so At the same time the Ct values in the sample table are updated Note Due to the further spread of the early exponential area of the product accumulation curves a logarithmic representation is bet
51. ate file RT Settings file rts in the qPCRsoft standard folder Saves a template file RTSettings file rts in any user selected folder Saves a project file RTProject file rtp in the qPCRsoft standard folder Saves a project file RT Project file rtp in any user selected folder Opens an analysis file RT Analyses File rta Saves an analysis file RTAnalyses File rta Closes a template or a project Closes all open projects or templates Prints a project Closes the software Reverses the last text modification up to 10 steps Restores the last deleted text item up to 10 steps Cuts a marked text area Copies an active and or marked text area Pastes atext area copied to the clipboard Deletes an active and or marked text area Marks a complete text area Opens the window for creating user profiles and changing the password Opens the window for managing digital signatures of projects Switches the project explorer view in the main window on or off Switches the toolbar view in the main window on or off Opens the window for creating files for spectral color compensation Resets the connected device to the initial state Activates the connected device Opens the window for configuring the system with color filter modules Opens the window for general basic software settings Arranges project windows horizontally Arranges project windows vertically qPCRsoft CA
52. ation Fig 63 Project window for the AACt method 07 2012 71 Evaluation e Parameter settings e Display of the fluorescence curves for the target gene and the reference gene e lf standards have been defined display of the standard curve and the validation curve for the expression level of the gene of interest in relation to the reference gene and the calculated factors e Display of the sample table with the added values 5 4 2 Parameter settings for the AACt method ddCt Quantification 72 Gene of Interest GOT FAM c mye hr Group Threshold Group 1 39 125 Reference genes ROX Tubulin 3 Fig 64 Parameter settings for the AACt method The following parameters must be set for the AACt method Option SELECTION LIST GENE OF INTEREST GOI REFERENCE GENES GROUP THRESHOLD Description Selection of an evaluation created for the experiment Selection list of combinations of measured dyes and target genes to be quantified Only one target gene at a time can be selected Selection list for the reference genes In contrast to the target gene several reference genes can be selected at the same time The number of list sheets in the display range therefore grows with each reference gene With the L amp I symbol you can remove all selected reference genes from the selection If several experiments were carried out on the PCR plate select the group of the experiment to be analyze
53. aved standard curve Create new evaluation Delete evaluation Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold for detecting Ct values Import a saved standard curve qPCRsoft Software DELTADELTACT ADD AACT QUANTIFICATION DELTE AACT QUANTIFICATION OPTIONSAACT QUANTIFICATIONG AUTOMA THRESHOLD MELTING CURVE ADD MELTING CURVE DELTE MELTING CURVE OPTIONSMELTING CURVE AUTOMA THRESHOLD GENOTYPIN ADD GENOTYPING DELETE GENOTYPING OPTIONS GENOTYPING AUTOM THRESHOLD 1 4 2 Overview of the tools in the toolbar Create new evaluation Delete evaluation Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold for detecting Ct values Create new evaluation Delete evaluation Opens a window for basic evaluation settings Determine threshold automatically Create new evaluation Delete evaluation Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold for detecting Ct values The buttons in the toolbar are mostly context sensitive The program automatically adjusts the toolbar to the window content and adds buttons provided that this is required and useful for the current project window view Buttons not accessible for the current contents of the workspace are hidden View toolbar You can display or hide the toolbar via the VIEW TOOLBAR menu command
54. basic assumption of the Livak method equal efficiency when amplifying the gene of interest and the reference gene is rarely the case in practice and the calculation can therefore lead to incorrect values The calculation method is selected in the DDCT OPTIONS window ro O Click on the amp symbol in the toolbar Alternatively call up the DELTADELTACT DDCT QUANT OPTIONS menu command Q Select the desired calculation method For the Pfaffl method the efficiency values can be determined automatically from the standard curves dilution series for the gene of interest and the reference gene if standards have been defined or the values can be entered manually in the respective fields ddCt Options Smoothing Scaling Ii none linear Without effidency calculation Livak method T Points logarithmic E With effidency calculation Pfaff method Calculate efficiencies from standards Baseline correction For all samples From cyde To cyde 3 15 Input efficiencies Sample specific Crop first cydes 5 Autom threshold Standard deviation of base lines Defined standards Displaying the validation curves and values To calculate the AACt values there is no need to determine a validation curve However it might be useful to consult it when checking the data quality Prerequisite for creating a validation curve is measuring a standard series with different dilution levels of target gene and
55. by mode activation after the PCR is complete Table Step 1 of 53 Lid temp c fio fv p S lrupe ctr fio Standby 7 Fig 7 Program header of the PCR program example for qTower settings Option List Description LID TEMP Sets the lid temperature The temperature of the heating lid should generally be slightly above the maximum block temperature to prevent liquids from evaporating from the reaction mixture and condensing at the walls or lid of the reaction cups PREHEAT LID If activated this option preheats the lid to the set LID TEMPERATURE before the actual PCR program starts This is the recommended standard setting to ensure the formation of a homogeneously tempered air cushion between the sample containers This leads to an improved temperature uniformity between the samples While the lid is being heated the block is kept at a constant 25 C If the option is deactivated the PCR program starts while the lid is still being heated Adjustable lid temperature 30 110 C Note After the heating lid has reached the target temperature this is followed by a 40 second equilibration before the program is started qPCRsoft 07 2012 21 Settings for a Real Time PCR experiment List temperature regulation of the thermoblock for ql ower Select block type for T Optical Device selection for qTower 2 0 and 2 2 BLOCK_CTRL The block temperature is regulated according to the selected tempera
56. d Section Defining groups p 41 Manually adjust threshold values The threshold value must be between 1 and 100 depending on the standardized representation of the fluorescence curves dRn Note The threshold value can be calculated automatically or set in the graph Opens the selection window with display options Setting the threshold value To determine Ct values for the evaluation a threshold value needs to be determined for each experiment first You have several options for setting the threshold value In the general options see section Making basic settings p 54 e Manually in the parameters for the respective evaluation see table above e Graphically in the fluorescence curves representation By having it calculated automatically 07 2012 qPCRsoft DeltaDeltaCt Autom Threshold Evaluation LJ Inthe chart move the black threshold line up or down with the cursor Press and hold the left mouse button while doing so At the same time the Ct values in the sample table are updated Note Due to the further spread of the early exponential area of the product accumulation curves a logarithmic representation is better suited for setting the threshold manually in the display range than a linear representation Q The automatic calculation of the threshold value is activated by clicking on the symbol Optionally you can call up the DELTADELTACT AUTOM THRESHOLD menu command Whether you
57. d but is also possible manually in the display area gt Parameter settings for genotyping on page 85 In addition to the fluorescence curves representation the results can also be displayed as a scatter plot or bar graph This can be selected via the respective tabs below the display area Ct Mutant Wild type FAM Mutant ROx Scatter plot End point Fig 85 Scatter plot for genotyping The scatter plot is divided into four quadrants for mutant heterozygote wild type and error The samples are assigned to one of the quadrants based on the 88 07 2012 qPCRsoft Evaluation measured relative fluorescence or the Ct values of the two dyes The respective cutoff value for the sample assignment is represented by two black lines in the scatter plot view To change the position of the lines and thus change the cutoff value select the lines and keep the left mouse button pressed Alternatively the respective cutoff value for mutant and wild type can also be entered in the respective fields in the selection area gt Parameter settings for genotyping on page 85 100 T3 dRn 50 Wild type FAM Mutant ROX Scatter plot End point Fig 86 Bar graph for genotyping The bar graph shows the measured relative fluorescences as bars The x axis is scaled from A to H based on the rows of the block i e the first twelve samples correspond to positions A1 to A12 in the block the next twelve samples correspond to
58. e By programming increments decrements the temperature or hold time can be modified by a specific amount from one cycle to the other within a loop This technology is for instance used for the Touch Down PCR 24 07 2012 qPCRsoft Settings for a Real Time PCR experiment a e a a ee eel Soke ee lees lt Fig 11 Entering increments for temperature and hold time Q Enter the desired changes for the temperature step the values of which you wish to modify within the loop Use the sign to specify a decrement i e temperature or hold time are reduced from cycle to cycle by the specified amount No sign or mark a decrement with the result that the parameter increases by the specified amount from cycle to cycle LI To modify the target temperature in steps enter the changes in the AT C column Q To modify the hold temperature in steps enter the changes in the AT S column Note The modified step must be within a loop Otherwise the entries in the columns AT C and AT S have no effect Note The extension of the hold time of one step affects the total run time of a protocol A program with many cycles and significant hold time increases takes substantially longer than a comparable program without a programmed extension Arranging a fluorescence measurement LI To define the measurement of the sample fluorescence in a temperature step of the PCR protocol click on the SCAN column of the temperature step A gr
59. e p 34 DEFINE SCAN REGION Sample measurement according to manual settings gt MANUALLY see section Manually defining the scan region p 31 The information on position excitation channel and detection and the selection list of the available dyes cannot be modified in this table Modifications of these general settings can only be made in the EDIT COLOR MODULES window gt see section Editing color modules p 92 Set the following parameters for each channel you wish to make a measurement for 1 Select the dye to be measured in the DYE column Click in the cell and mark the dye in the list that opens up Note The number of measured dyes does not have an influence on the scan time 2 Set the signal quality in the GAIN column The standard setting is 3 3 Activate the fluorescence measurement in the channel in the Measurement column by placing a green diamond Channels that are not marked with a diamond will not be measured 4 If necessary activate the reference dye measurement by placing a v in the PASS REF column 5 Enter the number of repetitions for the fluorescence measurements in the MEAS REPEATS field The standard setting is 3 Note An increased number of repeat measurements reduces the measurement value distribution but also creates longer scanning times and thus longer protocolling times 6 Select one of the options for the SCAN REGION MANUALLYOR ACCORDING TO LAYOUT 3 3
60. e range limits for the correction can also be adjusted in the project window 4 2 2 Adjusting the view in the MONITORING project window 48 In the Monitoring project window you can adjust the preset parameters MONITORING DISPLAY OPTIONS menu command for the display of the scaling as well as the range limits for the baseline correction 1 Click on the button above the chart A selection field for setting the display options and entering the baseline parameters opens 07 2012 qPCRsoft Monitoring FOR ALL SAMPLES baseline SAMPLE SPECIFIC baseline correction correction parameter parameter Fig 37 Parameter settings in the MONITORING project window 2 Change the baseline correction limits and activate the LINEAR or LOGARITHMIC option for the desired view of the fluorescence curves 4 2 3 Displaying and hiding measurement results for individual wells qPCRsoft The sample view in the MONITORING project window is controlled via the SAMPLES menu item in the project explorer Measurement results in the individual wells can be hidden or shown Note The selection in the project explorer only influences the display of the fluorescence data but not the measurement E Samples 12 3 4 5 6 7 amp 3 10 ii 12 wOeeGGececee EILE Q2 eeee00 Fig 38 Project explorer SAMPLES window The marking of the sample assignment corresponds to the marking on the SAMPLES project tab Sample type SymbolDefinition EMPTY Describe
61. eecceeececceeecceecceesceeccaeeceneceneeass 7 1 4 2 Overview of the tools in the toolbar cccccceccseececeeeeseeeeaeeeeaes 10 1 4 3 Project explorer components cccsececeececeeeeceeeeseeeeseeeseeeesaeeees 12 1 4 4 Project interface with project WINdOW cccccseceseeeeseeeeseeeeeeeees 13 2 Managing projects rc 15 2 1 Creating a new project or opening a Proje ct cc eeceeeeeeeeeeee es 15 Creating a NEW PLOjeCt ccccsecccsecccesceceeecceeecseeceueeseueeseeesseeessas 15 Creating a new project based on a template cccccceeeeeeeees 15 Opening a saved PLOjC ccccecccseccceeeeceeeeeeeesaeeeseeeeseeeeseeeeseeeeaes 15 Opening an automatically Saved project cccccecceeeeeseeeeeeeeeaes 15 Viewing the Projects ccccscccseecseeseeeseeeseeeceeceecoseeceeeceeenenenans 16 2 2 SAVING a CCIM PLAC errorae ay aca a ee eae 16 2 3 SAVING a OO CE sies ences a a aa eens tosets AEN 16 2 4 Importing Exporting ANALYSES cece seceeseeeeeeeeeeeeeseeeeseeeeaeeesaeeees 16 2 5 Closing project WINdOWS ccccceececeececeeeeeeesaeeeseeeeseeeeseeeeaaeeeaes 17 2 6 PANUNG iiit NE NA aa aeansaal 17 2 1 SIGNING DOECO 18 3 Settings for a Real Time PCR experiment cccceeeeeeeeeeeeees 19 3 1 Entering general information on the project ccccccceeeeeeeeees 19 32 Creating APCR protocol cccccseccceseecseeeceeeccueeceusecueesseees
62. eeesees 20 3 2 1 Editing Au GIN PlOlOCO lista Se e 21 3 2 2 Entering information in the program header cccccceseeeeeeeees 21 3 2 3 Entering the PCR protocol cccccccseecceeeceeeeceeeeseeeeceeeseaeeseeess 22 Inserting a new temperature step deleting a temperature step 23 Entering the target temperature hold time and heating cooling MCS AAE EEE gaits eee Secs A E eck meee er cei meee ETA 23 DOMINO TOOD S eaen at estet a cect A E aaesterset 24 Entering increments decrements for temperature and hold time 24 Arranging a fluorescence MEASUreEMENL cceeeccseeeeeeeeeeeeeeeees 25 Adding a melting curve sesiirveetat chant asictetursehictianeuubsdanseenierseaeians 25 Programming the temperature gradient TOptical 96 Gradient and TOWER 2 2 ONY keane ie on aaa 27 Programming a linear gradient TOptical 96 Gradient and TOWER 22 ONV esnie an a a a S 27 3 2 4 Graphical display and programming the PCR protocols 28 3 3 Defining the parameters for the fluorescence measurement 30 O23 Manually defining the SCAN region ccccceececeeeeeeeeeaeeeeeeeeeaeeees 31 3 3 2 Color compensation cccccccssceceseecececseecceeeceueeceueecueesseeseeeeseas 32 3 4 Editing the sample table cccccccsseccseeceeeeceeeeseeeceeeseeeesseeens 34 3 4 1 Defining Sample properties ccccceccceeecseeceeeeceeeeeeeeseeeesseeens 36 Entering sample properties in the layout
63. een diamond indicates that the measurements is active Fig 12 Defining a fluorescence measurement in the PCR run Define the parameters for the fluorescence measurement on the SCAN tab gt see section Defining the parameters for the fluorescence measurement p 30 Note If a step for melting curve determination is added the scanning process is automatically activated for this step For all other steps of the PCR protocol the allocation must be made manually Adding a melting curve Settings Thermocycler DNA melting project window qPCRsoft For experiments with intercalating dyes we recommend to check the specificity of the products by measuring a melting curve The device can be programmed to add the corresponding step in the PCR protocol Activate the option ACTIVE on the MELTING CURVE tab 07 2012 25 Settings for a Real Time PCR experiment 26 4 Steps scan __ c_ _mis__ goto eops_ fa ies oc ee ee eo DER hte EES Ee aEsA Eas ie oe ee ee ee eee S Fig 13 PCR protocol with a step for determining the melting curve Q A melting curve can be added to the program table by activating the option ACTIVE ON MELTING CURVE tab The melting curve is added to the last temperature step in the table Q Toremove a melting curve from the program table deactivate the option ACTIVE on melting curve tab LI Set the individual parameters of the melting curve step on the MELTINGCURVE tab Melting c
64. el Quant t DeltaDeltact gt Melt curve d b Melting curve Melt Temperatures 1 Gene of Interest GOD Group Threshold FAM Groupi 4 767 Fig 73 Parameter settings for melting curve quantification Set the following parameters for the meltings curve quantification Option Description SELECTION LIST Selection of a selection created for the experiment GENE OF Selection list of target gene dye combinations INTEREST GOI Generally an intercalating dye must be selected for the target gene for the melting curve analysis According to the selection the fluorescence and regression curves for the concentration are displayed GROUP If several experiments were carried out on the PCR plate select the group of the experiment to be analyzed Section Defining groups p 41 THRESHOLD Manually adjust threshold values The threshold is now effective on the DERIVATIVE tab Only curves whose maximum dRn dT is greater than the threshold are analyzed Note The threshold value can be calculated automatically or set manually in the chart gt see also Setting the threshold value below Opens the selection window with display options Setting the threshold value For the correct evaluation a threshold value must be determined for the melting curves You have several options for setting the threshold value e Inthe general options see section Making basic settings p 54 e Manually in the parameters for the
65. emperature the lid heating mode and the thermocycler s block type The programming table offers a clear representation of the individual steps of the program The THERMOCYCLER tab contains three list sheets The tabs for switching between the list sheets are located at the bottom of the window List sheet Function TABLE Contains a table for programming PCR programs GRAPH Offers the option for graphical programming of PCR programs MELTING CURVE Is used to enter parameters for measuring a melting curve 07 2012 qPCRsoft Settings for a Real Time PCR experiment 3 2 1 Editing a PCR protocol Programs can be edited in the tabular or graphical representation You can switch between the two alternative representations via the TABLE and GRAPH screens To edit one step the current active step is highlighted light red you can use the corresponding function from the CYCLER menu in the menu bar or the corresponding symbol from the toolbar Symbol CYCLER menu Description command ca ADD EMPTY STEP Inserts a new step behind the active step aar DELETE STEP Deletes the active step Sto CUT STEP Cuts the active step l COPY STEP Copies the active step a PASTE STEP Inserts a copied step behind the active step 3 2 2 Entering information in the program header The program header contains several general device specific program settings e Options for lid heating e Temperature control method e Stand
66. ence curves dRn Note The threshold value can be calculated automatically or set manually in the chart gt see also Setting the threshold value below Opens the selection window with display options see Displaying the fluorescence curves for absolute quantification on page 60 When changing to the scatter plot or end point bar graph two fields are displayed for the CUTOFF value for the WILD TYPE and the mutant instead of the Threshold field Genotyping Wild type E Group Cutoff Wild type FAM ha Indude passive reference Group 1 35 55 Mutant Cutott Mutant ROX r oyu Fig 81 Parameter settings for the scatter plot and the bar graph of a genotyping qPCRsoft 07 2012 85 Evaluation Setting the threshold value To determine Ct values for the evaluation a threshold value must be determined for each experiment You have several options for setting the threshold value inthe general options see Making basic settings on page 54 manually in the parameters for the respective evaluation see table above graphically in the fluorescence curves representation by having it calculated automatically LI Inthe chart move the black threshold line up or down with the cursor Press and hold the left mouse button while doing so At the same time the Ct values in the sample table are updated Note The logarithmic view is more suitable for placing the threshold manually in the display area than the li
67. entration Table Standard curve Validation Fig 69 Validation curves view for the AACt method Probentyp Gen GOI Ct GOI Ct Ref Gen dct Ref cen RQ GOI RQRef gen Norm Expre amp y 7 oh emye aa 0 13 42 Unbekannt c myc 25 32 2 96 0 13 0 1286 43 Unbekannt GAPDH 18 44 0 1 0 1286 44 Unbekannt GAPDH 20 38 0 1 0 1286 A5 Unbekannt c myc 29 34 9 03 0 0 001 Unbekannt c myc 31 43 9 03 0 0 001 Unbekannt GAPDH 17 44 0 98 1 97 0 001 Unbekannt GAPDH 19 43 0 98 1 97 0 001 Ag Unbekannt c myc 26 32 5 97 0 02 0 0314 410 Unbekannt c myc 28 33 5 97 0 02 0 0314 All Unbekannt GAPDH 19 43 0 97 0 51 0 0314 412 Unbekannt GAPDH 21 35 0 97 0 51 0 0314 BS Kalibrator c myc 20 38 0 1 1 B6 Kalibrator c myc 22 33 0 1 1 B7 Kalibrator GAPDH 18 44 0 1 1 1 BS Kalibrator GAPDH 20 38 0 1 4 Tabelle Standardkurve Validierung Fig 70 Sample table view for the AACt method The selection of the displayed columns can be user defined Right click on a column header to display the selection field with following options Column Meaning WELL Position of sample COLOR OF CURVE Each sample is automatically assigned an unchangeable color which is used to display the corresponding fluorescence curve SAMPLE NAME Name of sample 07 2012 qPCRsoft qPCRsoft Evaluation SAMPLE TYPE Type of sample GROUP Assignment of the sample to an experimental group GOI Gene of interest REFERENCE GENE Reference ge
68. ereich ermittelt Die untere und obere Bereichsgrenze sind in den Feldern VON ZYKLUS und BIS ZYKLUS einzustellen At this correction the base line is determined for every sample in the same area The lower and upper area limit has to be edited in the fields FROM CYCLE and TO CYCLE SAMPLE SPECIFIC This correction should be chosen if the curves have very different Ct values The lower area limit for the determination of the base line will adjusted in the field CROP FIRST CYCLES for all samples The upper area limit is separately found out by an algorithm for each sample Note The manner of the base line correction can be selected only in this dialog The area limits can however be adapted for the correction in the project window Calculation of the threshold as a deviation of x times of the standard deviation of the baselines factor can be adjusted under EXTRAS OPTIONS ANALYSIS in the main menu or based on defined standards with the goal to get the maximum value for the coefficient of determination R 07 2012 55 Evaluation FILTER Digital filter for smoothing the fluorescence curves Adjustable in steps SLIGHT MEDIUM and STRONG More setting options may be available depending on the analysis method used They are explained separately in the respective sections All items displayed on the ANALYSIS project tab can also be accessed quickly via the settings area of the baseline and displayed as linear or logarithmic represen
69. file in the list Click EDIT 4 Change the data in the USER PROFILE window Note If changing the password is allowed in the profile users with general limited rights can open their profiles in USER USER MANAGEMENT and change their passwords They do not have access to other settings 7 2 3 Deleting a user profile qPCRsoft 1 Click on the EDIT USER MANAGEMENT menu command to open the USER PROFILES window 2 Mark the user profile in the list 3 Click DELETE The user profile is deleted 07 2012 97 Index 8 98 Index A Absolute quantification create evaluation 59 delete evaluation 64 display fluorescence curves 61 edit parameters 60 import standard curve 64 results 62 standard curve 62 threshold value 60 AbsQuant menu Add abs quantification 59 Autom Threshold 61 Import standard curve 64 Options abs quantification 55 B Baseline correction 55 Block type 22 C Color compensation 32 Color modules 94 Cycler menu Add empty step 21 23 Copy step 21 Cut step 21 Delete step 21 23 Past step 21 D Data format 96 DeltaDeltaCt menu Add ddCt quantification 72 Autom Threshold 74 Delete ddCt quantification 79 Options ddCt quantification 55 76 Device initialization 94 E Edit menu Signatures 18 Undo Redo Copy Cut Insert Mark all Delete 19 User management 98 End point bar graph 91 Evaluation 54 Absolute quantification seeAbsolute quantification baseline correction
70. he commands offered in the toolbar may change according to context Project explorer 3 In the project explorer a drop down menu provides a quick overview of the most important information on the current open project Project interface 4 The project interface is used to process projects As soon as a new project is created or an existing project is loaded a window opens in which all relevant settings for the respective project can be made 1 4 1 Menu commands overview The menu bar is context sensitive and is automatically adapted to the program tasks Menu items that are not necessary for the current work interface are automatically hidden The following menu commands are available in the qPCRsoft software Menu Function Description FILE NEW Opens a new project OPEN TEMPLATE Opens a template RTSettings file rts qPCRsoft 07 2012 7 qPCRsoft Software EDIT VIEW SCAN EXTRAS WINDOW OPEN PROJECT OPEN AUTOM SAVED PROJECT SAVE TEMPLATE SAVE TEMPLATE AS SAVE PROJECT SAVE PROJECT AS IMPORT ANALYSES EXPORT ANALYSES CLOSE CLOSE ALL PRINT EXIT UNDO REDO CUT COPY PASTE DELETE MARK ALL USER MANAGEMENT SIGNATURES PROJECT EXPLORER TOOLBAR SET COLOR COMPENSATION DEVICE INITIALIZATION DEVICE IDENTIFCATION EDIT COLOR MODULES OPTIONS TILE HORZ TILE VERT 07 2012 Opens a project RTProject file rtp Opens an automatically saved project Saves a templ
71. he results of a relative QUGMUPICAUON sssansssicscnveswicds vvcesensanastcsiukadsecoubtensaanncussadaauexdonedcanneuees 67 3 0 0 Importing the standard curve for relative quantification 69 5 3 6 Deleting the evaluation of a relative quantification 08 70 5 4 AACGEMEOd urtasi a a ciate a 71 5 4 1 Creating a new evaluation for a AACt method ccceceeeee ees 71 5 4 2 Parameter settings for the AACt method cccccecceeeeeeeeeeeeees 72 5 4 3 Displaying the fluorescence curves for the AACt method 73 5 4 4 Setting the calculation mode for the standardized expression 14 5 4 5 Displaying the validation curves and values ccccccceee sees eeeee 15 5 4 6 Deleting a AACt method evaluation ccccceccseeceeeeeeeeseeeeeeeees 78 5 5 Meiing curve analysis seeria a a ai 79 5 5 1 Creating a new melting Curve analysis ccceecceeeceeeeeeeeeeeeees 79 5 9 2 Parameter settings for melting Curve analysis ccseceeeeeees 80 5 9 3 Displaying fluorescence curves melting Curves cccccseeee scenes 81 5 5 4 Displaying the sample table for the melting curves 008 82 5 9 5 Deleting a melting curve analysis cceecceeeeseeeeeeeaeeeneeeseeeneeeees 83 5 6 CSC MOLY DING arnap a E a a aR 84 5 6 1 Creating a new evaluation for genotyping ccceeceeeeeeeeeeeeeeees 84 5 6 2 Parameter settings for genotyping
72. he temperatures in the individual columns of the block are summarized in a table below the bar graph Gradient Step 1 of 4 View Margin ka Temp Row 12 Ee C Temp Row 0i As 7 C 64 8 65 0 65 6 66 5 67 5 68 5 69 5 70 5 71 5 72 4 73 0 73 2 Row 1 2 3 415 16 7 8 9 10 fi 12 Table Graph Gradient Melting curve Fig 16 Gradient tab with MARGIN gradient function LI Adjust the gradient progression by entering temperature values for the first TEMP Row 01 and last TEMP Row 12 column of the block The values displayed in the table are then updated LJ Alternatively you can click on the top end of column 1 or column 12 and change the height of the column with the left mouse button depressed Moving the columns changes the respective temperature value accordingly Programming a linear gradient TOptical 96 Gradient and qrOWER 2 2 only In addition to the option of defining a gradient by entering temperature values for columns 1 and 12 the gradient can also be programmed starting with an annealing temperature in the center of the block using fixed temperature increments LJ On the GRADIENT tab select the LINEAR option from the VIEW list LJ Enter the temperature for column 6 in the ANNEALING TEMP field and the desired temperature change from column to column in the INCREMENT field The values displayed in the table are updated after the values have been entered
73. ield with following options Column Meaning WELL Position of sample COLOR OF Each sample is automatically assigned an unchangeable color CURVE which is used to display the corresponding fluorescence curve SAMPLE Name of sample NAME 82 07 2012 qPCRsoft Evaluation SAMPLE Type of sample TYPE GROUP Assignment of the sample to an experimental group GENE Name of gene measured in the sample TM Melting temperature of the sample MEAN TM Mean melting temperature of replicates The individual columns can be shown or hidden by selecting or deselecting them Moreover the columns can be rearranged freely To exchange to columns press and hold the left mouse button and drag a column header to the desired location The display of the results in the table can thus be customized individually Display in the project explorer A shortened representation of the melting curves calculated by the software is displayed in the project explorer under Melt Curve This representation shows the graphical plot of the first derivative of the fluorescence values against the temperature Ea Melt Curve Fig 78 Representation of the melting curve derivatives in the project explorer 5 9 9 Deleting a melting curve analysis Melting curve Delete melting curve qPCRsoft A melting curve analysis that is no longer required can be removed 1 Activate the evaluation by selecting its description in the evaluation li
74. ing melting curves nnannnnnennanonnnnnnnnonnonrrnrrnrrnrrnrrnrenrrnnne 53 Evaluatio Merio 54 5 1 General functions in the evaluation project window 06 54 ora Making basic SettlngS ccccccsecceecceeecceeceeeeceeeceeeseeeseeeseeeseeeees 54 S12 Activating deactivating samples for evaluation ccceeeee ees 56 5 1 3 Exporting evaluation data ccccccccccccceeceseeeeeeeeseeeeseeeeseeeeaeeeaes 57 5 2 Absolute quantification si3ecccside02bie eeerieiaes stiisudeciianl Gdeetaeenels 58 5 2 1 Creating an evaluation for an absolute quantification 58 5 2 2 Parameter settings for absolute quantification cceeeeeeee sees 59 5 2 3 Displaying the fluorescence curves for absolute quantification 60 5 2 4 Displaying the standard curve and results of an absolute QUANITITIG AMON sages san ctehs toes E S 61 5 2 9 Importing the standard curve ccc ccecc cece eeeeeeeseeeee esse esse eesaeesaeenes 63 5 2 6 Deleting the evaluation of an absolute quantification 63 5 3 Relative Quantification cccccccsecccseeccseeceeeeceeeeceecceeeseueesueenaaes 64 5 3 1 Creating a new evaluation for a relative quantification 64 5 3 2 Parameter settings for relative quantification cccccceeeeeeeeees 65 5 3 3 Displaying the fluorescence curves in the relative quantification 66 5 3 4 Displaying the standard curves and t
75. ith the parameter settings measurement results and evaluations is created in the project window Opening an automatically saved project File Open autom The qPCRsoft programs enables you to automatically save the last completed saved project Real Time PCR run to a folder of your choice and thus prevents data loss due to unexpected terminations of a PCR run The terminated measurement can be recovered with the FILE OPEN AUTOM SAVED PROJECT menu command and saved under a different name as a project file qPCRsoft 07 2012 15 Managing projects To change the storage location of the file proceed as follows 1 Select EXTRAS OPTIONS to open the window of the same name 2 Open the GENERAL tab 3 Click and select a storage location The AUTOM SAVE option enables you to switch the automatic save function on and off Viewing the projects Window You can open several projects at a time Each project is displayed in its own project window With the commands from the WINDOW menu the project windows can be arranged horizontally vertically or overlapping 2 2 Saving a template All basic information required for performing an experiment that is stored in the project window on the SETTINGS tab such as the description of the experiment the PCR protocol scan settings of the optical system and the plate assignment can be saved as a template File Save 1 Call up the FILE SAVE TEMPLATE AS command template as
76. jects 2 Managing projects The qPCRsoft software saves all experiments in the project files A project contains different information required to perform a Real Time PCR experiment Description of the experiment e PCR protocol e Scan settings of the optical system e Plate assignment with detailed information on each sample e Measuring results and the corresponding evaluation after the experiment has been performed 2 1 Creating a new project or opening a project A project is always indicated in a project window in a section of the project interface of the main window Creating a new project File New To create a new project click on ial or call up the FILE NEW menu command A new project with standard presets is created in the project window Creating a new project based on a template A new project can be opened with saved presets template File Open gt a template 1 Click on or call up the FILE OPEN TEMPLATE menu command 2 Inthe standard window select the desired template to open files RT Settings file rts and confirm the selection with OK A new project with the parameter settings of the template is created in the project window Opening a saved project File Open Ei S project 1 Click on or call up the FILE OPEN PROJECT menu command 2 Inthe standard window select the desired project to open files RTProject file rtp and confirm the selection with OK The project w
77. ld value The threshold value must be between 1 and 100 depending on the standardized representation of the fluorescence curves dRn Note The threshold value can be calculated automatically or set manually in the chart gt see also Setting the threshold value below Opens the selection window with display options see section Displaying the fluorescence curves for absolute quantification p 60 Setting the threshold value To determine Ct values for the evaluation a threshold value needs to be determined for each experiment first You have several options for setting the threshold value In the general options see section Making basic settings p 54 Manually in the parameters for the respective evaluation see table above Graphically in the fluorescence curves representation By automatic calculation LJ Inthe chart move the black threshold line up or down with the cursor Press and hold the left mouse button while doing so At the same time the Ct values in the sample table are updated Note Due to the further spread of the early exponential area of the product accumulation curves a logarithmic representation is better suited for setting the threshold manually in the display range than a linear representation 07 2012 59 Evaluation LI The automatic calculation of the threshold value is activated by clicking on the symbol Alternatively you can call up the ABSQUANT AUTOM
78. le is mounted in the device If necessary you can also add dye names if these have not yet been added to the list Click on ACCEPT Repeat this for each color module you have installed If you wish to define a new color module that is not included in the list of available color modules click ADD A new color module with the description COLOR 000 000 00 0 is created You can define the properties on the right side of the dialog To delete a color module from the list mark the module with the cursor and click on DELETE To change the properties of a color module proceed as follows Activate the PROPERTIES checkbox Inthe POSITION list select the position of the color module on the carrier in the fluorescence probe Enter the code noted on the color module in the CODE OF MODULE entry field Enter the dye that is detected with the color module Click on The dye is added to the list below You can remove a dye by marking it in the list and clicking on Click ACCEPT to assign the properties to the marked color module Note A dye can only be assigned to one module If it is to be measured with a different module it first has to be removed from the first module 6 5 Connecting the device to the PC Extras Device identification The software gPCRsoft automatically recognizes which instrument is connected and whether it is switched on or not The instrument may also be switched on and off du
79. n MELT Additionally a symbol of an arrow pointing upwards is displayed with the melting curve steps Note The number of repetitions in melting curve steps cannot be modified Q The temperature value at each step can be modified using the mouse For this purpose press and hold the left mouse button to move the blue line of the temperature curve up or down qPCRsoft 07 2012 29 Settings for a Real Time PCR experiment 3 3 Defining the parameters for the fluorescence measurement Settings Scan project window 30 The product accumulation is measured by means of the increase in fluorescence in the Real Time PCR The following measurement parameters must be defined for that purpose e Dyes to be measured e Temperature step of the PCR protocol during which a measurement is to take place e The area on the PCR plate that is to be scanned The colors to be measured are defined in the SETTINGS SCAN project window Ei Real time pcr project demo_qTOWER rtp iol x settings Monitoring Analysis General Thermocycler Scan Samples 1 Blue 470n0mi 520n0ni FAM 3 oP 2 Green 515mm 545mm JOE 3 a 3 Yellow 535mm s 0nm HE 3 ae Meas repeats 3 Color compensation CFF f Scan region according layout Define scan region manually Fig 20 Project window with settings for the fluorescence measurement The SCAN tab contains a table with different parameters for defining the scan properties Up t
80. ne CT GOI Ct value of gene of interest CT REF GENE Ct value of reference gene MEAN CT GOI Mean Ct value of replicates of the gene of interest MEAN CT REF GENE Mean Ct value of replicates of the reference gene STD DEV CTGOI Standard deviation of the Ct values between replicates of the gene of interest STD DEV CT Standard deviation of the Ct values between replicates of the REF GENE reference gene CV CT GOI Variation coefficient of the Ct values between replicates of the gene of interest CV CT REF GENE Variation coefficient of the Ct values between replicates of the reference gene DCT GOI Delta Ct value for replicates of the gene of interest DCT REF GENE Delta Ct value for replicates of the reference gene RQ GOI Calculated relative amount for replicates of the gene of interest in the original sample RQ REF GENE Calculated relative amount for replicates of the reference gene in the original sample MEAN RQ Average calculated relative amount for replicates of the REF GENE reference gene in the original sample NORM EXPRESSION Standardized relative x fold expression level of the gene of interest in the sample in relation to the calibrator The individual columns can be shown or hidden by selecting or deselecting them Moreover the columns can be rearranged freely To exchange to columns press and hold the left mouse button and drag a column header to the desired location The display of the results in the table can thus be cust
81. near view because of the wider spread of the early exponential area of the product accumulation curves Genotyping LJ To activate the automatic calculation of the threshold value click on the a Autom threshold symbol Alternatively you can call up the GENOTYPING AUTOM THRESHOLD menu command Whether you choose manual or automatic calculation the resulting threshold value is updated and displayed synchronously in the THRESHOLD input field 5 6 3 Specifying genotyping options Special evaluation options are available for genotyping ay O Click on cM in the toolbar to open the GENOTYPING OPTIONS window Alternatively you can call up the GENOTYPING GENOTYPING OPTIONS menu command 86 07 2012 qPCRsoft Geno typing options Smoothing Ei none linear Points logarithmic Baseline correction For all samples From cyde To cyde 3 al 45 Sample specific Crop first cydes 5 Autom threshold Standard deviation of base lines Defined standards Cyde of interest Set to last cyde Set to cyde Descriptions If wild type wild type If mutant mutant If heterozygote heterozygote otherwise error Scatter Plot D based on intensities dAn based on Ct values Evaluation Fig 82 GENOTYPING OPTIONS window for genotyping settings Option Description CYCLE OF Select the cycle for evaluation This can preferably be the last cycle INTEREST end point
82. ngs Montorra BH anavas E C General ie Thermal Cyder Scan Samples q p Edit layout Create groups Unit ng qPCRsoft 07 2012 45 Monitoring 4 Monitoring All functions required for starting and monitoring a Real Time PCR run are combined on the MONITORING project tab 4 1 Starting the PCR protocol Activate the MONITORING tab in the project window to display the symbols for start ing the PCR and the MONITORING menu protocol defined in the SETTINGS project window in the tool bar Symbol MONITORING menu command gt START QPCR RUN La STOP QPCR RUN HH PAUSE QPCR RUN j i Ay VIEW OPTIONS hs Description Start the PCR run The PCR run is interrupted and will not be continued The data recorded up to this point is saved and can be evaluated The PCR run is interrupted The symbol flashes during the break The PCR run can be continued by clicking al again QTower only Opens and closes the thermoblock Defines default settings for the Monitoring view Warning Contamination of the thermoblock Do not leave the thermoblock open unless absolutely necessary There is a risk that the thermoblock becomes contaminated This will lead to a poorer quality and efficiency of the optical fluorescence measurement 46 07 2012 qPCRsoft 4 2 Monitoring project window q gPCRsoft Monitoring Display options for monitoring The MONITORING project window is divided into
83. nterest in relation to one or more reference genes often housekeeping enes If one of the samples is denoted as the calibrator the expression level of that sample is set to unity and the relative expression levels of all the other samples are given in relation to the calibrator sample For relative quantification standard dilution series are required for the gene of interest as well as for the reference genes 5 3 1 Creating a new evaluation for a relative quantification RelQuant Add rel quantification 64 1 Go to the ANALYSIS RELQUANT project tab If the tab is not visible click on the arrows 4 t in the tab bar This will scroll the tabs pat 2 Click onthe symbol in the toolbar Alternatively call up the RELQUANT ADD REL QUANTIFICATIONMenu command 3 An input window appears Enter the description for the current evaluation On the REL QUANT tab the following information is activated Real time per project 3Toptical_Demo_RelQuantl_1 rtp o mE UD Settings Monitoring l 4 Analysis 4d b E Abs Quant Ea Rel Quant ft DeltaDeltact Hi Melt curve d p Gene of Interest GOT Group Threshold Indude passive reference Group 1 1 189 gt Reference genes FAM GAPDH a wel a rane B6 Empty Group 1 a B7 W sta Standard Group 1 c myc 25 BS B si Standard Group 1 cmyc 26 Bg Bs Standard Group 1 c myc 26 B10 Empty Group 1 Bi B Empty Group 1 ha 4 lool I Table Stand
84. nterest in relation to one or more reference genes often housekeeping genes One of the samples has to be denoted as the calibrator The expression level ofthe calibrator is set to unity and the relative expression levels of all the other samples are given in relation to the calibrator sample In AACt method there is no need to measure standard dilution series However if the AACt method shall be validated within the same PCR run standard dilution series must be defined 5 4 1 Creating a new evaluation for a AACt method DeltaDeltaCt Add AACt Quantification qPCRsoft 1 Goto the ANALYSIS RELQUANT project tab If the tab is not visible click on the arrows 4 t in the tab bar This will scroll the tabs ddt 2 Click on the eg symbol in the toolbar Alternatively call up the DELTADELTACT ADD DDCT QUANTIFICATION menu command 3 An input window appears Enter the description for the current evaluation On the DELTADELTACT tab the following information is activated Real time per project 3Toptical Demo_ddCt_WithStandards1_1 rt E amp Settings Monitoring BS Analysis db _ fig Abs Quant Ea Rel Quant Ve DeltaDeltact Melt curve db ddCt Quantification Gene of Interest GOT Group Threshold Groupi 2 326 Reference genes FAM GAPDH x i sai esa Unknown Group 1 GAPDH PE Unknown Group 1 camyc 27 AB Saz Unknown Group 1 camyc 29 F 4 lool b Table Standard curve Valid
85. o four qT OWER or six TOptical qTOWER 2 0 2 2 color channels with different excitation and detection wavelengths can be used for the fluorescence measurement The parameters of the fluorescence measurement apply for all layout samples on which a measurement Is to be performed Parameter Option Description Pos Color module position in the device CHANNEL Color channel description EXCITATION Excitation and detection wavelength of the color channel DETECTION DYE Define the dye to be measured for the corresponding channel in the table by means of a selection list GAIN Regulation of signal intensity The intensity can be adjusted in steps between 0 and 10 The higher the value the higher the fluorescence signal in the corresponding channel Standard value 3 MEASUREMENT Activates dye measurement An active measurement is indicated with a green diamond PASS REF The LED technology of the device does not require a passive reference If you wish to measure a reference dye anyway place a checkmark in this column 07 2012 qPCRsoft Settings for a Real Time PCR experiment MEAS REPEATS Enter the number of repetitions of the fluorescence measurement Possible values 1 to 16 COLOR Activate spectral compensation see section Spectral COMPENSATION compensation p 32 SCAN REGION Sample measurement according to the layout of the ACCORDING LAYOUT samples on the SAMPLEStab gt see section Editing the sample tabl
86. of melting curve analysis you can differentiate whether the reaction has caused the formation of a specific PCR product or whether unspecific by products such as primerdimers were produced 5 5 1 Creating a new melting curve analysis Melting curve Add Melting curve qPCRsoft 1 Goto the ANALYSIS MELT CURVE project tab If the tab is not visible click on the arrows 4 in the tab bar This will scroll the tabs Eos 2 Click onthe symbol in the toolbar Optionally call up the MELTING CURVE ADD MELTING CURVEmenu command 3 An input window appears Enter the description for the current evaluation On the MELTCURVE tab the following information is activated Real time per project 3Toptical_Demo_Sybr Meltl_1_TOptical rtp o ES E Settings Monitoring OM Analysis d pb fas Abs Quant Ea Rel Quant t DeltaDeltact gt Melt curve 4b Melting curve Melt Temperatures 1 ha Gene of Interest GOT Group Threshold Bi 5 Unknown Group 1 77 8 z7 B2 Unknown Group 1 T7 7 77 B3 Unknown Group 1 77 6 77 B4 Unknown Group 1 77 6 77 B5 Unknown Group 1 77 5 T7 B6 Smp Unknown Group 1 77 4 Pi hd j 07 2012 79 Evaluation e Parameter settings e Display of the fluorescence values as a function of the temperature and or its first derivative e Display of the sample table 5 5 2 Parameter settings for melting curve analysis Melting curve Autom Threshold 80 a Abs Quant Gg R
87. ollowing units ng ng ul ng ml pg ul copies copies ul copies ml mg ml U ul 1U ml or Click on Ea to assign the sample properties to the three wells Repeat steps 7 10 for the other three standards Use the following parameters Wells Sample name Conc FAM VIC C4 C6 Std 2 50 5 D1 D3 Std 3 10 1 D4 D6 Std 4 0 1 0 05 07 2012 39 Settings for a Real Time PCR experiment 40 As sample type select the STANDARD option Assign the genes to the dyes as described in step 6 v The plate layout for a multiplex assay is complete Entering a sample layout for a singleplex assay The following example shows the definition of four samples and four standards with three repeat measurements each in the layout The GAPDH and c myc genes are analyzed with the FAM dye with the help of two sensors The FAM dye was selected on the SETTINGS SCAN project tab and activated for the measurement The indicated sample names and standard concentrations serve as examples only 1 2 Activate the EDIT LAYOUT button Empty the plate layout to make sure that no unintentional entries remain Mark the complete plate by clicking on the gray button in the top left of the layout In the SAMPLE TYPE list select the option EMPTY Click on Mark the three wells A1 A3 Make the following settings Parameter Entered value Sample name Sample 1 Sample type Unknown FAM GAPDH Click on bo to assign the sample
88. omized individually Display in the project explorer A shortened representation of the validation curves calculated by the software is displayed in the project explorer under DELTADELTACT The image displays the graphical plot of the dCt V values against the logarithm of the sample concentration i DeltaDeltaCt 1 75 z La S Hep Se a 2b a 1G Log Std Konz Fig 71 Representation of the validation curves in the project explorer 07 2012 77 Evaluation 5 4 6 Deleting a AACt method evaluation An evaluation that is no longer required can be removed DeltaDeltaCt 1 Activate the evaluation by selecting its description in the evaluation list of the Delete ddCt method tab quantification dC 2 Click onthe symbol in the toolbar Optionally call up the DELTADELTACT DELETE DDCT QUANTIFICATION menu command The evaluation is removed 78 07 2012 qPCRsoft Evaluation 5 5 Melting curve analysis When performing a melting curve analysis the temperature in the reaction mixture is Increased successively until the PCR product is denatured The dissociation of the fragment in single strands will result in the release of a intercalating dye The associated reduction of the fluorescence intensity is measured and recorded by the device By forming the first derivative of the fluorescence curve you will get a peak that describes the melting point and the approximate concentration of the PCR fragment By means
89. on in the name field It is included in the selection list after pressing OK and will be displayed in the corresponding window Templates that are no longer in use can be deleted with DELETE 3 4 Editing the sample table Project window Settings Samples 34 The sample table defines which sample is in which positionof the block These details are required for using the evaluation functions of gPCRsoft Here a sample can be described by means of its properties such as name gene type concentration and dye Furthermore samples from different experimental approaches can be combined in groups The necessary entries can be made on the SAMPLES tab after pressing the EDIT LAYOUT button The corresponding window is divided into different sections 07 2012 qPCRsoft Settings for a Real Time PCR experiment Edit layout Create groups 3 4 Fig 24 Window for editing the sample table Range Function LAYOUT VIEW 1 Graphical display of the well assignment on the microplate EDIT AREA 3 Edit area for the sample properties e Sample name e Sample type e Concentration of standard samples e Allocation of the dye and the analyzed gene SAMPLE TABLE 2 Summary of the information on each sample DYES 4 Dyes and assigned genes for each sample Note The sample table can also be edited after the Real Time PCR run has been completed qPCRsoft 07 2012 35 Settings for a Real Time PCR experiment
90. on page 51 07 2012 47 Monitoring 4 2 1 Default settings for the Monitoring view Monitoring Display options For all views in the MONITORING project window it is generally possible to choose between linear and logarithmic scaling for the graphical display of the data The setting for the baseline correction can also be changed od LY Select the MONITORING DISPLAY OPTIONS menu command or click ore in the toolbar linear logarithmic 4 Baseline correction gt For all samples From cyde To cyde 3 15 F Sample specific Crop first cydes Fig 36 DISPLAY OPTIONS window for changing the default settings of the Monitoring view Parameter Description Option SMOOTHING Setting the smoothing conditions for the measured data SCALING scaling options for the data LINEAR or LOGARITHMIC BASELINE There are two options for the baseline correction CORRECTION FORALL SAMPLES If this option is selected the baseline for every sample in the same range is determined The upper and lower range limit must be set in the FROM CYCLE and TO CYCLE fields SAMPLE SPECIFIC Select this option if the curves have significantly different Ct values The lower range limit for determining the baseline is set in the CROP FIRST CYCLES field for all samples The upper range limit is determined separately for each sample by an algorithm Note The type of baseline correction can only be set in this dialog Th
91. or this purpose AbsQuant Import standard curve 1 Use the pi symbol in the toolbar to open the IMPORT STANDARD CURVE window Optionally you can call up the ABSQUANT IMPORT STANDARD CURVE menu command The mathematical equation that the standard curve is based on and the associated dye are each displayed in the list fields of the window 2 Select one of the import options from the IMPORT STANDARD CURVE window and make the corresponding entries Option IMPORT FROM THIS RUN IMPORT FROM SAVED RUN MANUAL INPUT Meaning Imports a standard curve from the current open project lf several standard curves are saved in one project all curves are displayed and you can make a selection Imports a standard curve from a saved project lf several standard curves have been saved select the corresponding curve from the list Standard curve coefficients are entered manually Enter the gradient and the intercept for the equation Ct gradient log conc intercept 5 2 6 Deleting the evaluation of an absolute quantification qPCRsoft An evaluation that is no longer required can be removed 1 Activate the evaluation by selecting its description in the evaluation list of the method tab 2 Click on Bien in the toolbar The evaluation is removed 07 2012 63 Evaluation 5 3 Relative quantification Relative quantitation allows for determination of the relative expression level of a g g ene of i
92. owed by the administrator Has the same rights as the administrator but with the following limitations Cannot create or edit users Can only overwrite data after a confirmation prompt Cannot create and edit color modules No access to the MEASUREMENT ANALYSIS DEVICE FILE and USER MANAGEMENT tabs in EXTRAS OPTIONS User with limited rights E E Can change his own password if allowed by the administrator Has the same rights as a user with general rights but with the following limitations FILE NEW menu command is blocked Cannot save project templates Can open prepared project templates and start measurements with them but cannot edit the template exception GENERAL tab SCAN EDIT COLOR COMPENSATION is blocked Can import prepared evaluations but cannot change the settings in the evaluation Cannot access the EXTRAS OPTIONS menu command 07 2012 95 Working with the user management Setting up an administrator After the program has been installed an administrator must be set up and an administrator password defined 1 Start gPCRsoft 2 Click OK in the LOGIN DIALOG without entering a password k x J Login Dialog User name Password 3 Define the administrator password in the LOGIN window neben Sie ein neues Kennwort ein und best tigen Sie es Kenmore Kennwort best tigen 4 Setup the user accounts If no user management is desired deactivate it in Extras Options User man
93. plate in the upper part of the projrct window If the layout table shall be edited by copy cut and paste this can be done by using the right hand mouse button and keeping the Ctr button pressed 5 Press the Ctr button and hold it pressed during the whole operation 6 Mark the lines to be copied or cut by left klicking the mouse and drawing it up or down 7 Press the right mouse button and select the desired function Fig 32 The standard samples in the wells B1 B2 and B3 are copied to the clipboard 8 Select the line by left clicking the mouse from which the copied samples shall be inserted By right clicking the mouse the paste menu appears Alz Empty e B B standard B2 sz standard B3 std Standard 4 Standard E Standard Empty Empty Fig 33 The selected standard samples including their properties are now copied to the wells B5 B6 and B7 Please note that caused by their identical sample name the standard Std2 is now a six fold replicate 3 4 5 Exporting or importing the layout in Excel 44 The layout can be exported or imported as an Excel file xls The exported data can be edited in Excel and then reimported Q Right click on the sample table A context menu with the IMPORT TABLE FROM EXCEL FILE XLS and EXPORT TABLE TO EXCEL FILE XLS menu commands opens Select the desired menu command 07 2012 qPCRsoft Settings for a Real Time PCR experiment Le setti
94. positions B1 to B12 etc The cutoff value is set by moving the red or blue horizontal line up or down with the left mouse button pressed Alternatively the respective cutoff value for mutant and wild type can also be entered in the respective fields in the selection area of this view gt Parameter settings for genotyping on page 85 The cutoff values are thresholds after which the question whether a sample shows a reaction is answered with Yes See REACTION WILD TYPE and REACTION MUTANT table columns 5 6 5 Display of the values for the genotyping evaluation The sample table for the genotyping combines all data and corresponding measurements for the samples The columns shown in the sample table differ depending on the tab selected in the display area The table for the fluorescence curves provides a summary which includes the measuring data of both dyes If the fluorescence intensity at the end point is evaluated the sample table for the scatter plot is the same as the one for the bar graph The data summarized in the sample table for the fluorescence curves however partly differ from the data in the scatter plot or bar graph evaluation The selection of columns displayed can be defined by the user Right click on a column header to display one of the following selection fields qPCRsoft 07 2012 89 Evaluation 90 Fluorescence curves Scatter plot or bar graph vo Well Well Color of curve Color of curve Sample n
95. project interface is initially empty when starting the program Only when a new project is created or a saved project and or a template is loaded the project window opens 07 2012 13 qPCRsoft Software 14 Ej Real time pcr project demo_qTOWER rtp ioj x Settings Monitoring Analysis General Thermocycler Scan Samples 0 500 1000 1500 00 2500 Time s Table Step 1 of 5 Lid temp C fiio cae Tae TUBE _ CTRL fio Standby isele e ms Taps Tee ead i Sa Eos eee se es so v so EAR Table Graph DNA melting melting Fig 3 View of a project window In the project window all parameters measuring data and evaluations for one PCR plate are combined The basic functions are arranged on the three main tabs on the top Tab Function SETTINGS Contains all functions required for defining Real Time PCR runs MONITORING Contains different tools for monitoring Real Time PCR runs EVALUATION Includes the evaluation algorithms implemented in the software for analyzing acquired data These three tabs are always visible The view of the project window changes depending on the selection of a function tab To indicate the tab the descriptions refer to the tabs and list sheets are listed in the order in which they were activated and divided by a slash similar to a menu command e g SETTINGS THERMOCYCLER TABLE 07 2012 qPCRsoft Managing pro
96. quantification r NEw 74 DELETE Creates a new evaluation Deletes an evaluation Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold value for Ct value determination Imports a saved standard curve Creates a new evaluation Deletes an evaluation Te OPTIONS one AUTOMATIC THRESHOLD z IMPORT STANDARD CURVE Evaluation relative quantification peP NEW EM ag DELETE EM 07 2012 11 qPCRsoft Software OPTIONS AUTOMATIC THRESHOLD IMPORT STANDARD CURVE Evaluation AACt analysis dd Ct k daCt cad T ddt NEw DELETE OPTIONS AUTOMATIC THRESHOLD Evaluation melting curve p pi T bill NEW DELETE OPTIONS AUTOMATIC THRESHOLD Evaluation Genotyping E gs E Ee NEW DELETE OPTIONS AUTOMATIC THRESHOLD 1 4 3 Project explorer components 12 Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold value for Ct value determination Imports a saved standard curve Creates a new evaluation Deletes an evaluation Opens a window for basic evaluation settings Automatic determination of the fluorescence threshold value for Ct value determination Creates a new evaluation Deletes the current evaluation Opens a window for basic settings for the evaluation Automatically determines the threshold Creates a new evaluation Deletes the current evaluation Opens a
97. r the reference gene REF GEN MEAN Conc GOI MEAN CONC REF GEN STD DEV CT GOI STD DEV REFGEN Concentration for the gene of interest determined from the standard curve on the basis of the mean Ct value Concentration for the reference gene determined from the standard curve on the basis of the mean Ct value Standard deviation of the Ct values between replicates of the gene of interest Standard deviation of the Ct values between replicates of the reference gene 07 2012 qPCRsoft Evaluation CV CT GOI Variation coefficient of the Ct values between replicates of the gene of interest CV CT REFGEN Variation coefficient of the Ct values between replicates of the reference gene RELATIVE CONC Relative x fold expression level of the gene of interest in relation to the reference gene NORM REL CONC Relative x fold expression level of the gene of interest in relation to the reference gene standardized to the expression of the calibrator if defined The individual columns can be shown or hidden by selecting or deselecting them Moreover the columns can be rearranged freely To exchange to columns press and hold the left mouse button and drag a column header to the desired location The display of the results in the table can thus be customized individually Display in the project explorer A shortened representation of the standard curves calculated by the software is displayed in the project explorer under RELAT
98. rd concentrations serve as examples only 1 Activate the EDIT LAYOUT button 2 Empty the plate layout to make sure that no unintentional entries remain Mark the complete plate by clicking on the gray button in the top left of the layout Inthe SAMPLE TYPE list select the option EMPTY Click on 38 07 2012 qPCRsoft qPCRsoft 6 Settings for a Real Time PCR experiment Mark the three wells A1 A3 Make the following settings Parameter Entered value SAMPLE NAME Sample 1 SAMPLE TYPE Unknown FAM GAPDH VIC c myc Note The genes are allocated to the corresponding dye by entering the name of the gene or by selecting it from the displayed list in the TARGET table Click on ma to assign the sample properties to the three wells Repeat steps 3 5 for the other samples Use the following parameters Wells Sample name Sample type FAM VIC A4 A6 Sample 2 Unknown GAPDH c myc B1 B3 Sample 3 Unknown GAPDH c myc B4 B6 Sample 4 Unknown GAPDH c myc Defining the standard samples T 8 10 11 Mark the three wells C1 C3 Make the following settings Parameter Entered value Sample name Std 1 Sample type Standard Make the following entries in the TARGET table Dye Gene Conc FAM GAPDH 100 VIC c myc 50 Note The CONc column in the TARGET table is only available for the STANDARD sample type From the UNIT list choose a concentration or mass unit You can select from the f
99. reference gene If standard series have been measured for the target and reference gene the expression ratio between target and reference gene is represented graphically in the VALIDATION CURVE display For this purpose the mean Ct value of the target gene is subtracted from the mean Ct value of the reference gene for the corresponding dilution level and the resulting dCt V value is plotted against the logarithm of the concentration In the value range on the right the following calculated data is displayed 07 2012 15 Evaluation 76 e The coefficient of determination R e The gradient of the validation straight line e the intersection of the curve with the y axis at x 0 offset The gradient of the curve should not exceed a value of 0 1 The assumption then applies that the efficiencies of the amplification of the gene of interest and the reference gene are more or less identical and the calculation of the AACt values produces valid data R3 Slope Offset 0 53 Log Std Conc Table Standard curve Validation Fig 68 Validation curves view for the AACt method You can switch between the AACt calculation views using the Table Standard curve and Validation tabs The validation curves and the values are automatically calculated by the qPCRsoft software and updated if the settings change RAM c myc FAM GAPDH Re 39996 0 99921 Slope 3 59 4 04 Offset 15 62 20 03 PCR Efficiency 0 81 0 77 Log Konz
100. respective evaluation see table above e Graphically in the representation of the fluorescence curves derivative e Automatic calculation LJ Inthe chartDERIVATIVE move the black threshold line up or down with the cursor Press and hold the left mouse button while doing so At the same time the Tm values in the sample table are updated LJ The automatic calculation of the threshold value is activated by clicking on the tall symbol 07 2012 qPCRsoft Evaluation Optionally you can call up the MELTING CURVE AUTOM THRESHOLD menu command Whether you choose manual or automatic calculation the resulting threshold value is updated and displayed synchronously in the corresponding THRESHOLDinput field 5 5 3 Displaying fluorescence curves melting curves In the display area the measured fluorescence curves are shown in relation to the temperature and are either standardized to the highest fluorescence value or both standardized to a target value of 100 depending on the settings made in the MELT CURVE OPTIONS window The data is displayed as a linear or logarithmic representation depending on the selected display option a Melt Curve Options Smoothing Scaling E none linear a Points logarithmic Baseline correction From cyde 5 Standard deviation of base lines Defined standards Scaling All curves start at 100 Maximum initial fluorescence 100 Fig 74 Display options for melting curve an
101. ring running qPCRsoft Whether an active connection is established will be indicated in the lower left corner of the status line If a connection cannot be obtained in between 30 seconds you can use the EXTRAS DEVICE identification in order to solve the problem 6 6 General settings in the qPCRsoft software Extras Options qPCRsoft General settings for the qPCRsoft program can be made in the OPTIONS window Note For most of the settings in the OPTIONS window you need to be logged into qPCRsoft as an administrator Select the EXTRAS OPTIONS menu command to open the window of the same name Make the following settings on the tabs 07 2012 93 Functions in Extras menu Tab Function GENERAL Define that the last measurement results are saved automatically gt Opening an automatically saved project on page 15 DATA FORMAT Select decimal separator for the data export LANGUAGE Select language of the gPCRsoft program interface MEASUREMENT SENSITIVITY Set the basic sensitivity for optical measurements MEAS REP COLOR COMP Enter the measurement repetitions for recording the color compensation SHOW NEGATIVE VALUES RESULTING FROM COLOR COMPENSATION If activated negative values are also displayed in the sequence of the color compensation otherwise the output is i Oia ANALYSIS You can enter a factor for the quantitative evaluations QUANT FACTOR for the melting curve analysis MELTING FACTOR and for
102. s an empty position on the PCR plate UNKNOWN Sample of unknown concentration or dilution measuring sample STANDARD Sample of known concentration or dilution CALIBRATOR Sample whose gene of interest expression level is set as 1 NO TEMPLATE CONTROL NTC POSITIVE CONTROL Complete reaction preparation but without matrix strand Positive control preparation for which a reaction product is expected AL JE JL JL Ji 07 2012 49 Monitoring Negative control preparation for which no NEGATIVE CONTROL reaction product is expected Active wells i e displayed wells are marked with their sample type symbol For deactivated wells the position is white and the fluorescence data is hidden Empty wells are marked E No data is saved for empty wells LJ Click with the mouse to switch The activation changes with each click on a well LJ You can change adjacent wells by holding the mouse button and moving the cursor over the wells Q Complete rows and columns can be inverted by clicking on the letter or number of the row A H or column 1 12 Q The complete plate can be switched by clicking into the empty field on the top left between A and 1 Q To activate all wells click on the symbol below the chart Q To activate only samples of a specific type click on the corresponding symbol below the chart To activate multiple sample types at the same time keep the Ctrl key pressed when clicking on the sample types
103. st of the method tab 2 Click on the Za symbol in the toolbar Alternatively call up the MELTING CURVE DELETE MELTING CURVE menu command The evaluation is removed 07 2012 83 Evaluation 5 6 Genotyping Genotyping serves to determine sequence differences between a sample and a standard The standard is defined as the reference sequence wild type the genetic condition of the sample is to be determined in the experiment Genotyping shows which alleles an individual has inherited from its parents 5 6 1 Creating a new evaluation for genotyping Genotyping Add 1 Goto the ANALYSIS GENOTYPING project tab genotyping If the tab is not visible click on the arrows 4 t in the tab bar This will scroll the tabs 2 Click on the a symbol in the toolbar Alternatively call up the GENOTYPING ADD GENOTYPING menu command 3 An input window appears Enter the description for the current evaluation The following information is activated on the GENOTYPING tab Real time per project 3Toptical Dermo_Genotypingl_1 rtp lo Si Settings Monitoring E Analysis a Ea Rel Quant St Deltadeltact W Melt curve Bi Genotyping ae Genotyping Genotyping1 2 je o i i Indude passive reference Group 1 30 539 gt Mutant Group 1 Unknown Group
104. t 77 min Fig 40 Status bar of the MONITORING project tab TABLE list sheet Lid 100 2 C Fig 41 Monitoring project window with PCR protocol table 07 2012 51 Monitoring Element Description LID Current lid temperature TEMPERATURE DISPLAY Current block temperature STEPS Temperature steps in the PCR protocol The active step is marked by a green arrow C Target temperature of the step M S Hold time format min s GRAPH list sheet 0i Fig 42 Monitoring project window with PCR protocol table The GRAPH list sheet contains the same elements of the graphical representation of the PCR protocol as the SETTINGS THERMOCYCLER GRAPH project window Once again the active step is marked by a green arrow T PROFILE list sheet E F F 50 a 0 0 500 1000 1500 2000 2500 Feit s Fig 43 Monitoring project window with T profile In the representation of the temperature profile a yellow progress bar indicates the step that is currently being performed 4 4 Displaying product accumulation curves 52 The product accumulation is documented by means of fluorescence measurements during the PCR run To display measurement curves for the product accumulation select the AMPLIFICATION or RAW DATA option from the VIEW list In the chart the fluorescence intensity I is plotted against the number of cycles in relative units The color of the curve that is being displayed corresponds to the color
105. t has currently been inserted To do so select the type from the devices list 3 2 3 Entering the PCR protocol The PCR program is entered into the program table of the TABLE list sheet One row of the table contains the parameters of a temperature step You can navigate in the program table by using the mouse or the four arrow keys lt gt A W on the keyboard Each entry is confirmed with the ENTER key or the gt arrow key The cursor jumps into the corresponding field in the adjoining column If the cursor is in the last row an additional temperature step is inserted with W 22 07 2012 qPCRsoft Settings for a Real Time PCR experiment Table 4 Step 1 of 5 Lid temp C fro Mi gt a TUBE CTRL f fi 0 Standby 7 elen x ms Tae Tes E E ee ee a sa ee so v eo he iri i Ea Ee Table Graph DMA melting Fig 8 Program table fora PCR The following values are entered into the table or calculated from the default values Value Description SCAN Measures the sample fluorescence during this step if marked STEPS Number of the step in the temperature program Is automatically numbered consecutively C Enter the target temperature of the step in C M S Enter the hold time of the target temperature GOTO LOOP Define the loop with the number of repetitions for a cycle AT C Enter the increment or decrement of the target temperature within the PCR run AT
106. tandard curve and the sample table via the CURVE and TABLE list sheets For the display of the standard curve the Ct values of the standard samples are plotted graphically against the logarithm of their concentration In the value range on the right the following calculated data is displayed e the coefficient of determination R2 of the linear equation e the standard curve gradient e the intersection of the curve with the y axis at x 0 offset e The PCR efficiency The standard curve and the values are automatically calculated by the qPCRsoft software and updated in case of settings modifications R Slope Offset PCR Effidency 0 86 o 05 1 15 2 35 3 Table Standard curve Fig 53 Standard curve for absolute quantification The sample table for the absolute quantification contains all data and the associated measurement values for the samples qPCRsoft 07 2012 61 Evaluation 62 B1 EE Standard Group 1 20 47 20 B2 B i2 Standard Group 1 20 58 20 B3 Std2 Standard Group 1 20 49 20 Boo Empty Group 1 Table Standard curve Fig 54 Sample table for absolute quantification The selection of the displayed columns can be user defined Right click on a column header to display the selection field with following options Column Description WELL Position of sample COLOR OF Each sample is automatically assigned an unchangeable color CURVE which is used to display the corresponding fluorescence curve SAMPLE NA
107. tations For this purpose a selection for display options can be opened in the corresponding window via the right arrow button FOR ALL SAMPLES baseline SAMPLE SPECIFIC baseline correction correction parameter parameter Fig 46 Individual setting of the baseline correction and fluorescence curve scaling during the evaluation 5 1 2 Activating deactivating samples for evaluation 56 Samples from individual wells can be activated or deactivated for evaluation in the project explorer SAMPLES menu item This enables you for example not to include excessive values when calculating mean values Note The selection in the project explorer only influences the analysis of the fluorescence data Measured data will not be deleted J Samples 2 eeee0 Fig 47 Project explorer SAMPLES window for de activating the samples in the evaluation The marking of the sample assignment corresponds to the marking on the SAMPLES project tab level is set as 1 NO TEMPLATE CONTROL NTC Complete reaction preparation but without matrix strand Sample type Symbol Definition EMPTY E Describes an empty position on the PCR plate UNKNOWN im Sample of unknown concentration or dilution measuring sample STANDARD 6 Sample of known concentration or dilution CALIBRATOR K Sample whose gene of interest expression 07 2012 qPCRsoft POSITIVE CONTROL NEGATIVE CONTROL Evaluation Positive control preparation for which a reaction prod
108. ter suited for setting the threshold manually in the display range than a linear representation LI The automatic calculation of the threshold value is activated by clicking on the ae symbol Alternatively you can call up THE RELQUANT AUTOM THRESHOLD menu command Whether you choose manual or automatic calculation the resulting threshold value is updated and displayed synchronously in the corresponding THRESHOLDinput field 5 3 3 Displaying the fluorescence curves in the relative quantification 66 In the display range the measured data standardized to the value 100 for highest fluorescence intensity is plotted against the cycle for the selected target gene The target gene dye and the reference gene dye combinations are each assigned a list sheet that can be activated by clicking on the gene dye tab on the bottom Since only one target gene dye combination is permitted at a time the fluorescence curves of the selected combination are displayed whenever a new combination is selected The number of the available list sheets depends on the number of the selected reference genes The fluorescence data is displayed as a linear or logarithmic representation depending on the selected display option For both display options the program shows brief information on the sample if the cursor is placed on one of the curves Switching the display options for the chart 1 Click on the button in the parameter bar A selection window
109. the genotyping GENOTYPING FACTOR in the list fields This factor will be used for the automatic calculation of the threshold DEVICE Enables you to define a file name into which the device communication data will be written The recorded data is used for error diagnosis Appendix FILE If activated the qPCRsoft program starts and the file opens if you select a file of this type in the file explorer of the operating system USER MANAGEMENT Activate or deactivate the user management If the user management is deactivated no login prompt will appear at the program start The functions for setting up the user management and for signing projects will not be available 94 07 2012 qPCRsoft 7 7 1 qPCRsoft Working with the user management Working with the user management Note on the general data security Due to the encryption used reading and changing project template evaluation and communication files generated by qPCRsoft is only possible with qPCRsoft User groups The user management enables you to assign users to three different user groups with different rights Administrator E E E E Has unlimited rights for all program functions Can create delete block and unblock users and assign rights to them Can change his own password and those of the other users Can deactivate the user management in EXTRAS OPTIONS USER MANAGEMENT User with general rights E E Can change his own password if all
110. tocols quickly In the SETTINGS THERMOCYCLER project window you can switch between the table view and the graphical mode via the TABLE or GRAPH sheets 28 07 2012 qPCRsoft Settings for a Real Time PCR experiment E Real time pcr project demo_qrOWER rtp q O x Settings Monitoring Analysis General Thermocycler Scan Samples 0 500 1000 1500 000 500 Time 5 Graphi Step 1 of 5 Table 4 Graph DNA melting Fig 19 Graphical programming mode The graphical programming is generally performed in the same way as in the programming table Q By selecting a step clicking on it it becomes active and is highlighted light red LI The bottom part of the display shows the number of the corresponding protocol step Next to it the number of repetitions in loops right and scanning processes middle can be programmed in this field The number of repetitions is indicated as a figure e g 40x and can be edited by clicking on it with the left mouse button Planned measurements are displayed by means of a green diamond in the middle of the field and can also be selected or deselected with the left mouse button LI Temperatures and hold times are indicated as numerical values above or below the blue line that displays the corresponding temperature level at the individual steps The values can be modified by clicking with the left mouse button Melting curve steps are marked with the additio
111. ture program Particularly if the heating and cooling rates are high and the hold times are short the actual sample temperature can differ from the desired temperature TUBE CTRL The sample temperature is calculated upfront based on the measured block temperature and is then regulated to that temperature This method is particularly recommended for fast protocols MAN_CTRL The manual block temperature control allows a very accurate matching of the experiment to the device thanks to the factor that can be adjusted between O and 10 The method for factor 0 is BLOCK_CTRL and for factor 10 TUBE_CTRL The installed block module is automatically detected by the TOptical thermocycler during device initialization with without gradient function When creating a new program this module is accepted as default However it is possible to create a program for a different block format than that currently installed for example a program with a temperature gradient although no block capable of a gradient has currently been inserted The desired block type can be selected from the list The installed qTOWER with 2 2 without gradient function 2 0 is automatically detected during initialization When creating a new program this type is accepted as default However it is possible to create a program for a different device type than that currently installed for example a program with a temperature gradient although no block capable of a gradien
112. uct is expected Negative control preparation for which no reaction product is expected Active wells i e wells included in the analysis are marked with their sample type symbol For deactivated wells the position is white and the fluorescence data is hidden Empty wells are marked E Q E Click with the mouse to switch The activation changes with each click on a well You can change adjacent wells by holding the mouse button and moving the cursor over the wells Complete rows and columns can be inverted by clicking on the letter or number of the row A H or column 1 12 The complete plate can be switched by clicking into the empty field on the top left between A and 1 To activate all wells click on the symbol below the chart To activate only samples of a specific type click on the corresponding symbol below the chart To activate multiple sample types at the same time keep the Ctrl key pressed when clicking on the sample types 5 1 3 Exporting evaluation data The data from the fluorescence measurement can be exported as csv files In addition the graphical display of the measurement results can be copied to the clipboard as a hardcopy and is hereby made available for other programs E E E E Right click on the graph A selection window for export and hardcopy appears Click on COPY CHART to copy the chart to the clipboard Select the SAVE CHART option to export the fluorescence data The SAVE AS
113. urve Step 1 of 5 Start temp C 60 Increment 4T 1 End temp C 95 Heating rate C s 5 Equilibration s 6 active Table Graph Melting curve Fig 14 DNA melting curve screen The following parameters can be modified Parameter Description START TEMP C Start temperature of the melting curve END TEMP C End temperature of the melting curve EQUILIBRATION S Time for equilibration of the sample in a temperature before a measurement is performed INCREMENT AT Difference between two adjoining temperature steps in C HEATING RATE Heating rate of the block C s ACTIVE Add a melting curve on the end of the PCR protocol The fluorescence measurement is automatically defined when the melting curve is recorded 07 2012 qPCRsoft qPCRsoft Settings for a Real Time PCR experiment Programming the temperature gradient TOptical 96 Gradient and qrOWER 2 2 only Gradients can be programmed over the whole temperature range of the thermoblock between 3 0 C and 99 C The gradient range can be max 40 C LI Gradients are defined in the program table by entering two temperature values separated by a dash The first value entered corresponds to the temperature in column 01 left block side the second value to the temperature in column 12 right block side Fig 15 Entering the temperature gradient The progression of the temperature gradient can be reviewed on the GRADIENT tab T
114. urve Table Fig 44 Evaluation project window The individual evaluation methods can be accessed via the subordinated tabs For each selection method different evaluations can be created Toolbar and menu commands are adjusted to the requirements of the selected method tab 5 1 General functions in the evaluation project window 5 1 1 Making basic settings Presets can be made for some evaluation parameters oh 1 Click on ac in the toolbar 2 Setup the following parameters 54 07 2012 qPCRsoft qPCRsoft Abs Quant Options Smoothing Scaling none linear 5z Ponts Clogatn Baseline correction C For all samples From cyde To cyde 3 15 F Sample specific Crop first cydes 5 kai Autom threshold Standard deviation of base lines Evaluation 0 Defined standards Fig 45 Option SMOOTHING SCALING BASELINE CORRECTION AUTOM THRESHOLD Window for basic evaluation settings Function Smoothing the fluorescence curves on the basis of the calculated moving average over a range of 2 to 12 measuring points or representation without smoothing Linear or logarithmic representation of fluorescence curves Bei der Korrektur der Basislinie ist zwischen zwei Optionen fur die Art der Korrektur zu wahlen At the correction of the base line you can choose between two options FORALL SAMPLES Bei dieser Korrektur wird die Basislinie fur jede Probe im gleichen B
115. ysis E General fi Thermal Cyder Scan E Samples db Analysis 1 zi B Unknown Analysis 1 5 B BB unknown Analysis 1 w B unknown Analysis1 m B unknown Analysis 2 a B unknown Analysis 2 B B unknown Analysis 2 o BB unknown Analysis 2 a E unknown Analysis 2 2 B Unknown Analysis 2 J B Unknown Analysis1 BB unknown Analysis 1 B Unknown Analysis1 not a i ao Fam Fig 30 Sample layout with four groups 42 07 2012 qPCRsoft Settings for a Real Time PCR experiment 3 4 3 Layout preview Samples Preview layout The layout preview provides a complete overview of the layout of the PCR plate with samples and the corresponding information that has been saved for the samples LJ Open the layout preview by clicking on the symbol in the toolbar Optionally call up the SAMPLES PREVIEW LAYOUT menu command The layout preview is displayed in the VIEW SAMPLE PLATE window The layout preview provides an overview of the following properties e Position on the PCR plate e Genes to be determined e Sample type by means of color marking at the edge e Underlined in color to indicate group affiliation If you move the cursor to a specific position all known settings for this position such as sample names sample type and group and all genes and dyes to be measured for the sample as well as the concentration in the case of standards are displayed in detail Fellkultur 4h Zellkultur 24h SSEAM GAPDH BA

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