User Manual
Contents
1. value must be equal to or higher than the baseline Start value b Determine whether you want to specify the threshold or allow the Pro Study software to auto calculate the value If you specify the Threshold value yourself you can use any numeric value above O zero Specifying Analysis Settings for your Study NOTE For more information on the auto baseline and auto threshold algorithms see the Prime Pro 48 Real Time PCR Operation Manual C Specify the desired Cq Variation value If a plate control well in your study has a Cq Variation value above the one you specify it is indicated by a D icon in the Well Table so you can exclude that well from the analysis if desired To exclude the well right click it in the Well Table and select Exclude 4 Click OK Pro Study re analyzes your data using the new analysis settings Reviewing the Data in your Study The data in your study can be viewed graphically or in a table on the Amplification Plot Melt Curve and Results tabs in the Pro Study software The example here shows the Amplification Plot tab for a Relative Quantification study containing three experiments 881218 8 3313 33133 Bes IEIEIEIFIEIEIEIFIEIKIEIE x 388833 PRR RRR RRR 3 38 8 8 8 9 5 85 85 8 8 5 8 9 3 88 88 8 9 8 8 8 S 8 8 8 zlsiaielejeioinizjejeijeisjelsigjeisialgiaieieieieieia AFTER TRIPS RN sella sisislelelgisisisicic slala EERE SE ERE 38183 31338 533388 S lssisisisis 283232 5 RRRAR RA2. your Study Amplification Plot The Amplification Plot displays the normalized or baseline subtracted fluorescence signal for a well at each cycle Using the Amplification Plot settings you can control how data are displayed in the Well Table and graph e Hover over a line in the Amplification Plot to highlight it in the Well Table e From the Experiments list select one or more experiments to limit what data appear in the graph and Well Table e From the Assay drop down menu select one or more assays to limit what data appear in the graph and Well Table e From the Plot Type drop down menu specify whether you want to show normalized R or baseline subtracted AR data in the graph e From the Graph Type drop down menu specify whether you want to show the data using a logarithmic or linear scale e Click drag the mouse over an area of the graph to zoom in on that area To clear the zoom and return to the full view of the graph click the Undo Zoom button a e Drag the threshold line for an assay up or down on the graph to change the analysis settings without opening the Analysis Settings dialog box The threshold can only be adjusted when viewing the data on a log scale e If you turned off the Auto Baseline option in the Analysis Settings dialog box you can drag the baseline start and end point gt indicators for an assay left or right on the graph to change the baseline settings x 0 048 Figure 4 Amplifica
3. 1 Click the Plate Layout tab 2 From the Experiments drop down menu select the experiment you want to use as the mother plate for your study Setting Up a Study o For Standard Curve studies the mother plate must have at least two wells with the role Standard but with different quantities For Relative Quantification studies any plate can be used as the mother plate However the mother plate must have at least one well that meets the criteria to serve as a plate control For more information on plate control requirements see Designating Plate Control Wells in your Study on page 7 3 Click the Mother Plate check box The previously designated mother plate is demoted to a be a daughter plate NOTE If you had a plate control well designated on the previous mother plate it is cleared and a new plate control will need to be assigned on the new mother plate For more information see Designating Plate Control Wells in your Study on page 7 Designating Plate Control Wells in your Study At least one well on the mother plate must be designated as the plate control Once assigned on the mother plate Pro Study automatically identifies wells containing the same assays reporter dyes and sample name on the other plates in your study During analysis the Pro Study software uses the Cq data from the plate control wells identified on each plate as a basis for comparing values across plates 1 Click the Plate Layout tab 2 From the Expe
4. N WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY 2014 Bibby Scientific All rights reserved Intended use Contents Introduction What is Pro Study Requirements for a standard curve study Requirements for a Relative Quantification Study Adding an Experiment Designating the Mother Plate Designating Plate Control Wells in your Study Specifying Analysis Settings for your Study Analyzing a Standard Curve Study Analyzing a Relative Quantification Study Reviewing the Data in your Study Well table Amplification plot Melt Curve Results Tab Standard Curve Relative Quantification Exporting Study Data to a Report or Presentation Technical Assistance The copyright of this instruction book is the property of Bibby Scientific This instruction book is supplied by Bibby Scientific on the express understanding that it is to be used solely for the purpose for which it is supplied It may not be copied used or disclosed to others in whole or part for any purpose except as authorised in writing by Bibby Scientific Bibby Scientific reserves the right to alter change or modify this document without prior notification Bibby Scientific Limited Beacon Road Stone Staffordshire ST15 OSA United Kingdom Tel 44 0 1785 812121 Fax 44 0 1785 810405 e mail technehelp bibby scientific com www techne com Page 5 5 5 5 6 6 7 00 10 10 11 12 12 12 13 13 14 Intro
5. Operation Manual RE This document and its contents are proprietary to Bibby Scientific and its affiliates Bibby Scientific and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Bibby Scientific Bibby Scientific does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY BIBBY SCIENTIFIC DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY PCRMAX IN CONNECTIO
6. R EEE AEAEE AAEE AEAEE AEAEE AEAEE AEAEE eee RRR TERR RR EE ae RRR Figure 1 New study Well Table The Well Table appears on the Amplification Plot Standard Curve Melt Curve and Results tabs The columns in the Well Table vary depending on your type of study Using Plate Layout Amplification Plot the Well Table settings you can control what data are A gt E included in the Amplification Plot Melt Curve and Ca Ski Deu Quantity Mean Qi su Der tr Baseline Stat Ba Results graphs 0 14 20000 20000 0 3 pa 0 14 20000 20000 0 m 0 12 5265 282 5079 958 e Select specific rows in the Well Table to display uE A the data in the graph 0 12 4967 596 5079 958 By default all rows are selected highlighted SS BER in yellow and so data for all wells in your study are shown in the graph e Hover over a row in the Well Table to highlight it in the graph e Specify filter criteria in the empty fields above each column to find data more easily In this example the Well Table is filtered to show only wells with a Cq Standard Deviation less than or equal to 0 15 Figure 2 Well table To clear the filter and return to the full data set click the Clear Filter button e Click a column heading to sort the Well Table by the data in that column e Select the Disable Auto Scroll button to stop the Well Table from scrolling vertically as you highlight data in the graph Reviewing the Data in
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8. dragging and dropping experiments onto the Pro Study software or by right clicking the Experiment list on the Amplification Plot Melt Curve or Results tab and selecting Add Experiment 2 Navigate to the ecod experiment data files you want to include in your study and click Open TIP If the experiment files are saved in the same folder you can Ctrl or Shift click to select multiple files for inclusion in your study at once 3 Repeat this procedure until all desired experiments are added to your study NOTE Pro Study automatically selects one experiment to serve as the mother plate The mother plate is the plate against which the other experiments in the study will be compared You can change this setting to designate any other experiment as the mother plate For more information see Designating the Mother Plate on page 6 NOTE If one or more of the experiments you selected is of a different type has a different thermal profile or has a different number of thermal cycles than the first experiment added to the study an error message opens for each unsupported experiment Click OK through each error message Pro Study will add any remaining supported experiments to your study Designating the Mother Plate Every study must have one experiment designated as the mother plate The mother plate is the plate against which the other experiments in the study will be compared The other experiments in the study are referred to as daughter plates
9. duction What is Pro Study The Pro Study software enables you to combine data from multiple Prime Pro 48 experiments into a study for analysis Currently only Standard Curve and Relative Quantification experiments are supported NOTE We do not recommend combining experiments from multiple instruments because differences in raw fluorescence values between instruments might increase the systematic error in your measurements Requirements for a Standard Curve Study To ensure successful analysis a Standard Curve study must contain o One or more experiments of the same type with the same thermal profile except for melt profile and number of thermal cycles o One mother plate if the study includes more than one experiment At least two standards with different values o One or more plate control wells in each plate Requirements for a Relative Quantification Study To ensure successful analysis a Relative Quantification study must contain One or more experiments of the same type with the same thermal profile except for melt profile and number of thermal cycles o One mother plate if the study includes more than one experiment o One or more plate control wells in each plate o One reference sample against which the target non reference samples will be analyzed o At least one target non reference assay Setting Up a Study Setting up a study is a three part process described in detail in the following section
10. e Analysis Settings Ell button 2 On the Cq Settings tab for each assay do the following a Determine whether you want to specify the baseline start and end values or allow the Pro Study software to auto calculate those values If you specify the baseline Start and End values yourself you can use any numeric value between 1 and the total number of cycles in your study The baseline End value must be equal to or higher than the baseline Start value b Determine whether you want to specify the threshold or allow the Pro Study software to auto calculate the value If you specify the Threshold value yourself you can use any numeric value above O zero NOTE For more information on the auto baseline and auto threshold algorithms see the Prime Pro 48 Real Time PCR System User Guide C Specify the desired Cq Variation value If a plate control well in your study has a Cq Variation value above the one you specify it is indicated by a Ba icon in the Well Table so you can exclude that well trom the analysis if desired To exclude the well right click it in the Well Table and select Exclude a Click OK Pro Study re analyzes your data using the new analysis settings Analyzing a Relative Quantification Study 1 Click the Analysis Settings 1 button 2 On the RQ Settings tab do the following a From the RQ Analysis Method drop down menu specify the desired analysis method Singleplex or Multiplex If your study includes only singleplexed we
11. ell Table Equation pe y 3 38x 34 31 0 994 Standard Curve Figure 5 Standard curve Reviewing the Data in your Study Relative Quantification RQvsS Relative Quantification studies generate a bar graph that includes error bars The displayed histogram can be toggled to group data by assay or by sample Exporting Study Data to a Report or Presentation ample Evaluauun Hale LUUY 14 DZ F Plate Layout Normalized Melt Thermal Profile Difference Melt You can export the data from your study in a variety PB Export Oplions of formats including comma and tab delimited text eee PA files Microsoft Excel spreadsheet and Microsoft Export Location H PowerPoint csv TSV Excel PowerPoint Export Options RU ss Ro D on oe 2 Type a name for the exported file MEM Dala The Export File Name field is pre populated with the name of the study or if you have not yet named the study with the name New Study 3 Click Browse and navigate to the folder where you want the exported data saved 4 Select the format in which you want your data exported 5 In the Export Options area select the check box for each type of data you want exported The options available here vary depending on the type of study you ran and the file format you chose to output If a type of data is not available it appears grayed out in the Export Options area In this example the Plate Layout Ther
12. lls the drop down menu only includes that one option If your study includes both singleplexed and multiplexed wells both options are available Only the wells matching your selection will be analyzed The other wells will be omitted from the analysis b From the RQ Algorithm drop down menu specify the desired normalization method AACg or Efficiency Correction The AACq method is supported only if you have a single reference gene and the amplification efficiencies are equal and at 100 If your study does not meet these requirements you must use the Efficiency Correction method For more information on these methods see the Prime Pro 48 Real Time PCR Operation Manual C From the Reference Sample drop down menu select one sample to be used as the reference sample d For each assay specify PCR Efficiency can be any numeric value between 0 200 Error can be any numeric value greater than or equal to O zero e Select the Reference Assay check box for every assay you want to use as a reference assay You must leave at least one assay unchecked to be used as the target assay 3 On the Cq Settings tab for each assay do the following a Determine whether you want to specify the baseline start and end values or allow the Pro Study software to auto calculate those values If you specify the baseline Start and End values yourself you can use any numeric value between 1 and the total number of cycles in your study The baseline End
13. mal Profile and Results Table are being exported to Excel Figure 7 Export options 6 Click OK Your exported data opens in the appropriate software application for the file format you chose to export Y Results Table u A rn vor Le u Technical Assistance For technical assistance go to www techne com or contact your local distributor MSDS Material safety data sheets MSDS are available on the Techne website at www techne com Product Documentation If you require additional product documentation you can obtain PDFs from the Techne website Go to www techne com or contact your local distributor Bibby Scientific Bibby Scientific Ltd Beacon Road Stone Staffordshire ST15 OSA United Kingdom Tel 44 0 1785 812121 Fax 44 0 1785 813748 e mail info bibby scientific com www bibby scientific com Bibby Scientific Middle East Ltd BPO Box 27842 Engomi 2433 Nicosia Cyprus Tel 357 22 660 423 Fax 357 22 660 424 e mail sales bibby scientificme com www bibby scientific com Bibby Scientific France Batiment le Deltaparc Silic Paris Nord 2 7 rue du Canal BP 55437 Villepinte 95944 ROISSY Charles de Gaulle France Tel 33 0 1 48 63 78 00 Fax 33 0 1 48 63 78 01 Email ventes bibby scientific fr www bibby scientific com Bibby Scientific US Ltd Bibby Scientific Asia Ltd 3 Terri Lane Suite 10 Room 607 Yen Sheng Centre Burlington NJ 08016 64 Hoi Yuen Road Kwun
14. riments drop down menu select the experiment designated as the mother plate for your study 3 Right click the desired well on the plate layout diagram and select Assign as Plate Control o For Standard Curve studies the well you designate as the plate control must have an assay with the role Standard Unknown or Positive as well as have a sample assigned Additionally the plate control well must not be excluded from the study For Relative Quantification studies the well you designate as the plate control must have an assay with the role Unknown or Positive as well as have a sample assigned Additionally the plate control well must not be excluded from the study Wells containing the same assays reporter dyes sample name and assay names are labeled Plate Control on the plate layout diagram for every experiment in your study On the other tabs in the Pro Study software plate control wells are indicated by the G icon in the Well Table Specifying Analysis Settings for your Study As you change settings in your study including adding experiments and changing the mother plate and plate control designations the Pro Study software automatically re analyzes your data using the default baseline threshold and Cq variation values If you want to use values other than the default settings you can specify so in the Analysis Settings dialog box as described in the following sections Analyzing a Standard Curve Study 1 Click th
15. s of this document o First you must add one or more experiments to a study The experiments must be of the same type Standard Curve or Relative Quantification have the same thermal profile except for melt profile and have the same number of thermal cycles o Next for studies containing more than one experiment you designate one experiment as the mother plate The mother plate is the plate against which the other experiments in the study will be compared Pro Study automatically selects one experiment to serve as the mother plate However you can change this setting to designate any other experiment as the mother plate o Finally you designate at least one well on the mother plate to serve as a plate control Once assigned on the mother plate Pro Study automatically identifies wells containing the same assays reporter dyes and sample name on the other plates in your study During analysis the Pro Study software uses the Cq data from the plate control wells identified on each plate as a basis for comparing values across plates Adding an Experiment When you start the Pro Study software it opens to a new blank study To analyze data from multiple experiments in the study you must add two or more Prime Pro 48 experiments of the same type Standard Curve or Relative Quantification with the same thermal profile except the melt profile and number of thermal cycles 1 Select File Add Experiment TIP You can also add experiments by
16. tion Plot Reviewing the Data in your Study Melt Curve The Melt Curve graph displays the fluorescence signal or derivative fluorescence signal for a well at every 0 3 C 0 54 F of melting Using the Melt Curve settings you can control how data are displayed in the Well Table and graph e Hover over a line in the Melt Curve graph to highlight it in the Well Table e From the Experiments list select one or more experiments to limit what data appear in the graph and Well Table e From the Assay drop down menu select one or more assays to limit what data appear in the graph and Well Table e From the Plot Type drop down menu specify whether you want to show Raw or Derivative Melt Derivative Melt data in the graph Figure 4 Derivative Melt e Click drag the mouse over an area of the graph to zoom in on that area To clear the zoom and return to the full view of the graph click the Undo Zoom button Results Tab The Results graph displays the results of your study in one of the following formats depending on the type of study you ran Standard Curve or Relative Quantification Standard Curve Standard Curve studies generate a standard curve line graph The slope PCR efficiency and standard error percentage and R of that curve appear in the graph s legend The standard curve Is always shown though you can decide which data points to include in the graph by selecting the appropriate rows in the W
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