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ABI PRISM 7000 Sequence Detection Systems Relative

1. 1 Select the Instrument tab By default the standard PCR conditions for the Fink acdsee Jj a Thermal Profile Auto Increment PCR step of the two step RT PCR method are eee oie displayed i F a Est Time Remaining hh mm r Temperature Sample Heat Sink Cover Block r Cycle Add Cycle Add Hold Add Step Sample Volume 50 ul HED Help IT Dissociation Protocol E00 C Time mm ss Step IV 9600 Emulation State 2 Verify that Save As 2x ave in ocuments i x If you are using two step RT PCR The _ pa 255 default thermal cycling conditions are set E History e Ifyou are using one step RT PCR You set the thermal cycling parameters as shown in Desktop Thermal Cycling Conditions for One Step My Computer RT PCR on page 25 ce The sample volume is set to 50 UL My Network P The 9600 Emulation check box is selected File name Lived gt Save as type SDS Documents sds bd Cancel i As 3 Select File gt Save As enter a name for the RQ Plate document then click Save 4 Load the plate into the instrument 5 Click Start As the instrument performs the PCR run it displays real time status information in the Instrument tab When the run is finished the Position plate status values and the buttons are grayed out Hia well A1 si a e upper Additionally the Analysis button is enabled P IE corner During the run the
2. Samples 0200000000008 2000000000080 2000000000080 00000000 O 0 OT Endogenous Controls GAPDH Notes 10 Relative Quantification Getting Started Guide for 7000 v1 1 Selecting the Sequence Detection Chemistry and Reagent Configuration About SDS Chemistries Selecting the Sequence Detection Chemistry and Reagent Configuration About SDS Applied Biosystems offers two types of chemistries that can be used to detect PCR Chemistries products on SDS instruments as explained in the following table For more information about SDS chemistries refer to the Sequence Detection Systems Chemistry Guide Chemistry Process TaqMan reagents or kits Description TaqMan reagent based chemistry uses a fluorogenic probe to enable detection of a specific PCR product as it accumulates during PCR cycles Advantages e Increases specificity with a probe Specific hybridization between probe and target generates fluorescent signal e Provides multiplex capability e Optimized assays available e Allows 5 nuclease assay to be carried out during PCR Polymerization FORWARD R REPORTER PRIMER QR PROBE Q 3 QUENCHER Qu REVERSE PRIMER Step 1 A reporter R anda quencher Q are attached to the 5 and 3 ends of a TaqMan probe Cleavage e Step 2 During each extension cycle the AmpliTaq Gold DNA polymerase cleaves the reporter dye from the probe 5 3 5
3. 7000 Sequence Detection System and provides an overview of the RQ Study workflow Designing an RQ Experiment Describes the required components of an RQ experiment and describes the set up of a sample RQ experiment Also provides a list of the materials and equipment required for RQ studies Performing Reverse Transcription Outlines the preferred methodology for reverse transcribing total RNA to cDNA and provides guidelines for RNA quality and starting amounts of total RNA Generating Data from RQ Plates Explains how data generated during the PCR process is captured in RQ Plate documents and provides information about analyzing RQ Plate data Performing an RQ Study Explains how to analyze data from one or more RQ Plate documents and to save the results in an RQ Study document Relative Quantification Getting Started Guide for 7000 v1 1 iii Contents Preface vil Safety and EMC Compliance Information iX Chapter 1 Introduction 1 About the 7000 SDS ANSTUMEN minre re nriran EDT EG 1 About Relative Quantification nanana aaaea ea eee eee ees 1 ADOL ROER DEFINE NES sree eea ER As ed be E EN A 2 About the Sample RQ Experiment aiee cee ee eens 3 Materials and Equipment aaa eee eee 4 Chapter 2 Designing an RQ Experiment 7 NO FEER ONE SETE SEE ES a My tanie sa see ora aavtan ees Saeed ecard Ede weve anata eee 7 Selecting the PCR Method seen eee eee ennen eee eee tees 7 Specifying
4. dye fluorescence as a function of cycle number You can use this plot 2 In the Data drop down list to identify and examine irregular select Delta Rn vs Cycle amplification and to manually set the threshold and baseline parameters for the run Ct vs Well Position 1 In the RQ Results panel The Ct vs Well Position plot Select een puncaHon displays threshold cycle C as a pone function of well position You can 2 In the Data drop down list use this plot to locate outliers from select Ct vs Well Position detector data sets see Omitting Samples from a Study on page 41 for more information Refer to Terms Used in Quantification Analysis on page 27 for definitions of R AR Cx and other terms used in quantification analysis Notes Relative Quantification Getting Started Guide for 7000 v1 1 37 Chapter 5 Performing an RQ Study Analyzing and Viewing the Results of the RQ Study Viewing Gene Expression Plots Gene Expression plots show the expression level or fold difference of the target sample relative to the calibrator as log of the 2 value For example if the bladder has a log10 relative quantification value of 5 relative to the kidney then that gene detector is expressed at a level five times higher in the bladder than in the kidney Note Because the calibrator is compared to itself the expression level for the calibrator is always 1 View Des
5. e Amount of time you want to spend optimizing and validating your experiment Amplifying target sequences and endogenous controls in separate reactions singleplex PCR requires less optimization and validation than multiplex PCR Among the factors to consider when doing multiplex PCR are primer limitation the relative abundance of the target and reference sequences the endogenous control must be more abundant than the targets and the number of targets in the study IMPORTANT As the number of gene targets increases the singleplex format is typically more effective than the multiplex format because less optimization is required e Requirement for high throughput performance Running multiple reactions in the same tube in a multiplex experiment increases throughput and reduces the effects of pipetting errors For more information about multiplex and singleplex PCR refer to the Sequence Detection Systems Chemistry Guide PN 430019 The singleplex PCR method was used in the sample experiment for the following reasons e Large number of targets to be amplified 23 genes plus one endogenous control e No requirement for optimization and validation for singleplex experiments Notes 8 Relative Quantification Getting Started Guide for 7000 v1 1 Nsa ar Ch sss ee ee ee Er See oe oe oo eo ee ee ae Specifying the Components of an RQ Experiment SS ED Specifying the Components of an RQ Experimen
6. Convert total RNA to cDNA using the High Capacity Have you converted CDNA Archive Kit the total RNA to cDNA Goto chapter 4 Generating Data eee een from RQ Plates Chapter 3 Performing Reverse Transcription Performing Reverse Transcription Workflow Performing ua Reverse Transcription RT PCR Methods Primer Extended on mRNA 5 Synthesis of 1st cDNA strand S Performing Reverse Transcription cDNA generation 1 Prepare total RNA samples 2 Use the manual method of the High Capacity CDNA Archive Kit to generate CDNA As discussed in Selecting the PCR Method on page 7 there are two RT PCR methods that you can choose from when performing RQ experiments one step RT PCR or two step RT PCR IMPORTANT Applied Biosystems recommends that you use the two step RT PCR method for RQ experiments This guide assumes that RQ experiments are designed using two step RT PCR Further only the recommended reagent configurations are documented For additional options refer to the Sequence Detection Systems Chemistry Guide Notes Relative Quantification Getting Started Guide for 7000 v1 1 15 Chapter 3 Performing Reverse Transcription Guidelines for Preparing RNA Guidelines for Preparing RNA Quality of RNA Ensure that the total RNA you use for RQ experiments is of reasonable quality Starting Amount of Total RNA e Its A 60 280 ratio should be greate
7. Ctrl P Component E 2 4 Rn Typically you export sample setup data for LER ER newly created and newly run plates other data i types are exported for existing plates IN D IL L Liver L Live L E L L Live L L L T T 2 Enter a file name for the export file Note The name of the dialog box depends on the type of data you want to export 3 Click Save File name SampleComponene xportFile My Network P Save as type Component Export File 7 Cancel Notes 30 Relative Quantification Getting Started Guide for 7000 v1 1 Exporting RQ Plate Documents Reanalyzing Data Notes Relative Quantification Getting Started Guide for 7000 v1 1 31 Have you have one or more RQ plate documents E Bladder sds Kidney sds EF Liver sds Chapte Generate data and save 4 it in RQ plate documents assay Fielative Quantification 5 Have you created RQ Create a new RQ study an RQ Study pages G document ocument Have you added plates to the study Add one or more plates page 4 46 the study Have you configured the analysis settings for the study Specify the analysis settings page 39 for the study Analyze the results of the RQ study by viewing the amplification or gene expression plates Chapter 5 Performing an RQ Study Performing an RQ Study Workflow Performing an RQ Study 1 Create an RQ Study docum
8. without requiring the exact copy number of the template Further the relative levels of templates in samples can be determined without the use of standard curves About RQ Experiments RQ Experiment Workflow RQ Studies with the 7000 v1 1 Instrument Notes This document uses the term RQ experiments to refer to the entire process of generating cDNA from RNA reverse transcription through analyzing RQ studies The RQ experiment workflow has several steps as shown in the following figure a Designing an RQ See Chapter 2 Experiment Performing Reverse See Chapter 3 Transcription Generating Data from See Chapter 4 RQ Plates Performing an RQ Study See Chapter 5 The RQ Study Add On software for the 7000 v1 1 SDS instrument enables you to perform RQ assays which calculate the relative quantification values from data generated during real time PCR Without the add on software the 7000 v1 1 SDS instrument can perform AQ AD and Plus Minus but not RQ assays Note For instructions on installing the RQ Study Software refer to Appendix B The data collection part of the assay is a single plate document called the RQ Plate Amplification data from PCR runs is stored with sample setup information on the plate Relative Quantification Getting Started Guide for 7000 v1 1 About the Sample RQ Experiment Description of the Sample RQ Experiment The data analysis part of the assay
9. 2 Designing an RQ Experiment Designing an RQ Experiment Workflow Designing Designing an RQ Experiment an RQ Experiment e Select the PCR method multiplex or singleplex e Designate the targets calibrator endogenous control and replicates e Select the SDS chemistry and reagent configuration e Choose the primers and probes Selecting the PCR Method Traditional PCR is performed as a singleplex reaction where a single primer pair is present in the reaction tube or well Only one target sequence or endogenous control can be amplified per reaction target sequences and endogenous controls cannot be amplified in the same tube In multiplex PCR two or more primer pairs are present in the reaction Each primer pair amplifies either a target sequence or an endogenous control The availability of multiple reporter dyes for TaqMan probes each with different emission wavelength maxima makes multiplex PCR possible Notes Relative Quantification Getting Started Guide for 7000 v1 1 7 Chapter 2 Designing an RQ Experiment Selecting the PCR Method Both methods give equivalent results for relative quantification experiments Which method to use depends on the e Type of chemistry you will be using to detect PCR products Singleplex PCR can use either SYBR Green or TaqMan reagent based chemistry Multiplex PCR can use only TaqMan chemistry
10. Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N fle Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices NT Indicates a potentially hazardous situation that if not avoided could result in death or serious injury N py elsag Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for Important each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to Applied Biosystems instruments see Safety Symbols on page xi Examples The following examples show the use of safety alert words IMPORTANT You must create a separate a Sample Entry Spreadsheet for each 96 well microtiter plate AN ozto The lamp is extremely hot Do not touch the lamp until it has cooled to room
11. Master Mix Create an RQ Plate document gt Generating Data from Create detectors RQ Plates Set thermal cycling parameters Save and run the RQ plate Analyze and view the results Before You Begin Calibrating the 7000 SDS instrument Preventing Contamination Notes Check that background and pure dye runs have been performed regularly to ensure optimal performance of the 7000 SDS instrument For more information about calibrating the 7000 SDS instrument refer to the Online Help for the 7000 SDS instrument PCR techniques require special laboratory practices to avoid false positive amplifications Kwok and Higuchi 1989 The high throughput and repetition of these techniques can lead to amplification of a single DNA molecule Saiki et al 1985 Mullis and Faloona 1987 Relative Quantification Getting Started Guide for 7000 v1 1 19 lt Chapter 4 Generating Data from RQ Plates Preparing the PCR Master Mix Follow these recommended general PCR practices e Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification e Change gloves whenever you suspect that they are contaminated Maintain separate areas dedicated equipment and supplies for Sample preparation and PCR setup PCR amplification and post PCR analysis Never bring
12. Strand Displacement Qo orao Or Step 1 continued when both dyes are attached to the probe reporter dye emission is quenched Polymerization Completed Q _ 5 5 SS eee 3 5 Step 3 Once separated from the quencher the reporter dye emits its characteristic fluorescence SYBR Green I reagents Description Uses SYBR Green dye a double stranded DNA binding dye to detect PCR products as they accumulate during PCR cycles Advantages e Reduces cost no probe needed e Amplifies all double stranded DNA e Yields a melting profile of distinct PCR runs e Increases sensitivity for detecting amplification products relative to product length Limitations Binds nonspecifically to all double stranded DNA sequences To avoid false positive signals check for nonspecific product formation using dissociation curve or gel analysis Step 1 The SYBR Green dye within the SYBR Master Mix immediately binds with all double stranded DNA present in the sample During PCR AmpliTag Gold DNA Polymerase amplifies each target The SYBR Green I dye then binds to each new copy of double stranded DNA Notes Relative Quantification Getting Started Guide for 7000 v1 1 11 Chapter 2 Designing an RQ Experiment Selecting the Sequence Detection Chemistry and Reagent Configuration About Reagent There are several TaqMan kits and SYBR Green I dye chemistry kits ava
13. When the software opens enter the Registration code on the CD jewel case cover Cancel if you do not have a registration code Remove the CD place it in its holder and store it in a safe place Checking After the software is installed check that the program files are in the following Program File locations Location e The default location for the 7000 SDS software program Notes C Program Files ABI Prism 7000 Prism7000 exe Relative Quantification Getting Started Guide for 7000 v1 1 49 Appendix B e The Start menu location for the 7000 SDS software shortcut Start Programs ABI Prism 7000 ABI Prism 7000 SDS Software IMPORTANT The installer program creates a link to the ABI PRISM 7000 SDS Software by placing a shortcut icon on the desktop during the installation Clicking the icon opens the program from the desktop without having to go through the Start menu Notes 50 Relative Quantification Getting Started Guide for 7000 v1 1 Numerics 5 nuclease assay 11 9600 Emulation Mode 26 A ABI PRISM 7000 Sequence Detection System 1 absolute quantification 2 Absolute Quantification AQ Assay 1 add on software for RQ studies 49 AIF See assay information files Allelic Discrimination AD Assay 1 AmpErase UNG 12 amplification plots Amplification Plot view for RQ plates 29 representative 27 types of amplification plots for RQ studies 37 Analysis Settings dialog box 35 Applied Biosystem
14. dye and quencher information and optionally the gene name or symbol for the sample name You can view the contents of AIFs in a spreadsheet program such as Microsoft Excel Notes 48 Relative Quantification Getting Started Guide for 7000 v1 1 Upgrading the 7000 Software with RQ Study Installing the RQ This section describes how to upgrade ABI PRISM 7000 SDS Software v1 1 to add the Study Software RQ Study v1 0 software The new software is installed in the ABI PRISM 7000 folder For a new installation you can install the RQ Study v1 0 software when you install the ABI PRISM 7000 SDS Software v1 1 Follow the instructions in the ABI Prism 7000 SDS Installation Guide Before you begin installation check that you have e The RQ Study v1 0 Installer CD e Installed the current ABI PRISM 7000 SDS Software v1 1 e Administrator privileges To install the RQ Study software i 6 Insert the RQ Study v1 0 Installer CD into the CD ROM drive The installer on the CD starts automatically Follow the onscreen instructions as the software installs the files When the Welcome window opens click Next The New Installation page opens to indicate that the installer is ready to perform a new installation Click Next again The wizard loads all the necessary files creates the ABI PRISM 7000 SDS Software shortcut on the desktop and registers your software When the installation is complete click Finish
15. for other detectors you want to screen Manual Ct Well Information SI Threshold 0 05 nple Summary 7 Plate Y Amplific Plot f Threshold Auto CT F 421692 t 42 ACVR2 0 401890 Auto 40 H CD30 0 307250 Aut F 1 00 u FLT4 352249 Suto GAPDH 415943 Auto GTF2E 1 uto STF 21 Auto HR4 10 200000 uto a KAN ui NCAM L h SFR tt Fine R2 Pla Well ample Dete T fe Omit iver Bg 079 E E iver B10 079 E iver B11 iver B12 4 E dn B9 y 282 lt idne B10 Kidney 2 4 431 282 E Kidney B11 Kidney CCR2 34 319 0 282 Bladder B10 Bladder Select the Omit Bladder B11 Bladder CCR2 36 000 0 621 check box adder B12 Bladder CCR2 37 031 0 621 4 Z Plate Y Amplification Plot E Threshold ito CT 1 0 421 ACVR 401 Cr a CD3D 0 307250 SF 21 0 20000 0 T4 249 tt PDH 445943 m TF25 1 m e F2l t HTR4 D Al 0 297613 Auto ae The outlier is PTGFR 0 350872 Auto 0 j removed during analysis nd pl D Task A Liver CCR2 Target 33 88 Kidney CCR2 Target 34 01 EZE Notes 42 Relative Quantification Getting Started Guide for 7000 v1 1 Exporting RQ Study Results 1 Select File gt Export gt Results then select the data type to export e Sample Sum
16. on a well to include it in the plot Ctrl click to include multiple wells Click and drag to include multiple adjacent wells 3 Select an individual detector or All all detectors in the Detector drop down list Yr Yd Yresuns Vo a ne Detector MME Note You can modify the graph settings for several of the result views as explained in Modifying Graph Settings on page 39 Reanalyzing Data Raw data fluorescence data spectra R values and well information sample name detector and detector task are saved in an RQ plate document If you decide to omit wells or change well information after a run has been completed you must reanalyze the data Note After the software analyzes data the Analyze button is disabled Whenever you change a setting that requires reanalysis the Analyze button is enabled Notes Relative Quantification Getting Started Guide for 7000 v1 1 29 Chapter 4 Generating Data from RQ Plates Exporting RQ Plate Documents Exporting RQ Plate Documents 1 Select File gt Export then select the data type to export Ctrl N e Sample Setup txt ctrl o e Calibration Data csv e Background Spectra csv Gtr 5 Save As Import Sample Setup x Sample Setup e Component csv SER Gatraton eta Print Preview Spectra il S Rn CSV Print
17. 0 100 Group Spacing 70 X 10 100 Gio p Spec nz onu applicable i Bar deh 100 Iv DBn FF Sanda Edara ka vin Bar e To set the graph scaling for post run data set the y axis of the graph to linear or log 2 Select Apply The system applies the changes made in the graphical settings box to the active plot 3 Select OK to close the Graph Settings dialog For more information about graph settings refer to the Online Help for the 7000 SDS instrument Notes Relative Quantification Getting Started Guide for 7000 v1 1 39 Reanalyzing an RQ Study Chapter 5 Performing an RQ Study Reanalyzing an RQ Study If you change any of the analysis settings you must reanalyze the data before you can view any results You can switch between the variations of the Amplification and Gene Expression plots without having to reanalyze the data Suppose you select Liver as the calibrator then perform an analysis Next you view the Amplification and Gene Expression plots If you then want to use Kidney or Bladder as the calibrator you need to reanalyze the data before viewing results Similarly if you want to change the baseline or threshold values the endogenous control the control type or the RQ Min Max parameters you need to reanalyze your data Liver as Calibrator Kidney as Calibrator Bladder as Calibrator Bladde
18. 0 a T E eee 41 Exporting RO Study RESUS lt 2 cog craw se i aren a G2 ab wal dew DE RR E A aa 2 43 References 45 Appendix A Creating Detectors 47 Appendix B Upgrading the 7000 Software with RQ Study 49 Index Back Cover Relative Quantification Getting Started Guide for 7000 v1 1 V vi Relative Quantification Getting Started Guide for 7000 v1 1 Preface How to Use This Guide Purpose of This This manual is written for principal investigators and laboratory staff who conduct Guide relative quantification studies for gene expression using the ABI PRISM 7000 Sequence Detection System 7000 SDS instrument Assumptions This guide assumes that you have e Familiarity with Microsoft Windows 2000 operating system e Knowledge of general techniques for handling DNA samples and preparing them for electrophoresis A general understanding of hard drives and data storage file transfers and copying and pasting If you want to integrate the ABI PRISM 7000 Sequence Detection System into your existing laboratory data flow system you need networking experience Text Conventions This guide uses the following conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix e A right arrow bracket gt separates successive commands you sel
19. 2 Spectra view 28 starting a run 26 templates 24 workflow 19 RQ Results panel 36 37 RQ Sample grid 36 RQ studies adding RQ plates 34 Amplification Plots 37 analysis settings 35 confidence level 35 control type 35 data types 43 definition 3 exporting data 43 Gene Expression plots 38 omitting samples from 41 orientation 38 overview 33 reanalyzing data 40 results 37 RQ Study documents 33 RQ Study Software 49 worklfow 33 RQ Study Add On software 2 RT PCR one step 12 25 two step 12 15 S safety x sample RQ experiment assay design 13 components 10 creating detectors 48 description 3 PCR master mix 21 PCR method 8 probes and primers 13 reagent configuration 12 reverse transcription 17 RQ Plate document example 24 RQ Study document example 36 SDS chemistry 12 SDS chemistries 11 Services and Support obtaining viii 2 settings graph 29 39 Setup tab 24 singleplex PCR 7 53 Index software RQ Study 49 workflow Spectra view 28 experiment design 7 reverse transcription 15 standard curves 2 RQ experiment overview 2 standards xvi RQ plates 19 starting an RQ plate run 26 RQ studies 33 study RQ See RQ studies workstation safety xv SYBR Green I dye chemistry 1 1 symbols xi T TaqMan chemistry 11 TaqMan Universal PCR Master Mix 20 target associating with detectors 22 24 definition 9 tasks See detector tasks Technical Communications contacting viii Technical Support contactin
20. AZARD Biological samples such as tissues body fluids and Biohazard blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx 01 29cfr1910a_01 html Additional information about biohazard guidelines is available at http www cdc gov Workstation Safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions Nexen MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows ade
21. Applied Biosystems Relative Quantification ABI Prism 7000 Sequence Detection System gt Applied AS Biosystems i em 7000 Sequence petection Syst Introduction Designing an RQ Experiment Performing Reverse Transcription Generating Data from RQ Plates Performing an RQ Study O Copyright 2003 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER PLEASE REFER TO THE ABI PrIsM 7000 SEQUENCE DETECTION SYSTEM USER S MANUAL FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION Trademarks Applied Biosystems MicroAmp Primer Express and VIC are registered trademarks of Applera Corporation or its subsidiaries in the US and or certain other countries AB Design ABI PRISM Applera Assays by Design Assays on Demand FAM and MultiScribe are trademarks of Applera Corporation or its subsidiaries in the US and or certain other countries AmpErase AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc SYBR is a registered trademark of Molecular Probes Inc Microsoft and Windows are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4346727 Rev A 9 2003 Relative Quantification Getting Started Guide for 7000 v1 1 Introduction Introduces concepts related to relative quantification RQ experiments using the ABI PRISM
22. Assay 1 preventing contamination 19 Primer Express Software 13 primers 13 21 probes 7 13 21 Relative Quantification Getting Started Guide for 7000 v1 1 R Rapid Assay Development Guidelines 13 21 reagent configurations 12 real time PCR assays 1 reference passive 24 27 relative quantification comparative method of calculation 3 definition 2 experiments See also RQ experiments 2 real time PCR 1 references 2 RQ plates 2 RQ studies 3 sample experiment 3 Relative Quantification RQ Assay replicates 9 reporter dye 27 results RQ Study analysis 37 Results tab 28 reverse transcription guidelines for preparing RNA 16 High Capacity cDNA Archive kit 16 thermal cycling parameters 16 worfklow 15 Rn vs Cycle view 37 Rn See normalized reporter RNA guidelines for preparing 16 ROX dye 24 RQ Detector grid 35 RQ experiments components 9 designing 7 generating data 19 PCR 19 probes and primers 13 reagent configuration 11 requirements 9 reverse transcription 15 RQ plates 19 RQ studies 33 SDS chemistries 11 workflow 2 RQ Main Study view 34 RQ plates adding to RQ studies 34 Amplification Plot view 29 analyzing 27 Component view 28 data types 30 definition 2 detectors creating 47 exporting data 30 importing sample information 24 Relative Quantification Getting Started Guide for 7000 v1 1 Index RQ plates continued overview 19 Plate view 28 reanalyzing data 29 results 28 RQ Plate documents 2
23. RNA sample bringing the total starting amount of RNA to 1 ug per 100 uL reaction The RNA was then converted to cDNA using the universal thermal cycling parameters for two step RT PCR as described in Thermal Cycling Parameters for RT on page 16 The cDNA was stored at 20 C for 24 hours Notes Relative Quantification Getting Started Guide for 7000 v1 1 17 Do you have cDNA Have you prepared the PCR master mix Have you created an RQ plate document Have the detectors for the experiment been added to the software Are the default thermal cycling conditions for PCR set Have you saved the data from the PCR run Yes Have you viewed the RQ plate data Chapter 4 Generating Data in RQ Plates page 20 page 22 page 23 page 25 page 26 page 28 Go to chapter 5 Performing an RQ Study Reverse transcribe total RNA to cDNA Prepare the PCR master mix as directed in the TaqMan Universal PCR Master Mix Protocol Create a new RQ plate document Create detectors Program the default PCR conditions or enter the thermal cycling conditions for one step RI PCR Save the data in the RQ plate document View the RQ plate data to confirm that the run was successful Generating Data from RQ Plates Workflow Generating Data from RQ Plates 1 Prepare the PCR Master Mix using TaqMan Universal PCR
24. TF2B 0284137 Auto GTF2I 0 320520 Auto HTR4 0 200000 Auto KAN 0 297613 Auto NCAMI 10 209008 Auto NFA 0174955 Auto 10350872 Good clustering of replicate data No outliers ACYRI 0 421692 Auto ACVR2 0 401890 Auto CCR2 0 055709 o 42 SER 055709 CD3D 0 307250 Auto FGF21 0 200000 Auto FLT4 0 352249 Auto GAPDH 0 415943 Auto GTF2B 0 284137 Auto GTF 0 320520 Auto HTR4 0 200000 Auto KAN 0297613 Auto NCAMI 0 209008 Auto NFA 0174955 Auto 0 350872 Auto Potential outlier Notes Relative Quantification Getting Started Guide for 7000 v1 1 41 Chapter 5 Performing an RQ Study Omitting Samples from a Study 6 Do one of the following TT se re En e If outliers are present select the Well om T a CCR2 TE Aut TEE 40 Information tab find the outlying sample oa hun and select the Omit check box for the GAPDH 041543 A sample ot e If outliers are not present go to step 7 Baca Saar aae 7 Repeat steps 5 and 6 to screen the remaining aol led doula replicate groups re ett lve lee r 8 Select Analysis gt Analyze to reanalyze the s e r e r r T Bladder 510 Bladder CCR2 34 037 0 621 a run without the outlying data Feo ADE ACA EM A al 9 Repeat steps 3 to 8
25. ab He He poate M 9600 Emulation Times and Temperatures Two step RT PCR HOLD HOLD For reference only RT is complete at this 1 RT Step point 10 min 25 C 120 min 37 C Initial Steps PCR Each of 40 cycles AmpErase UNG AmpliTag Gold DNA Melt Anneal Extend 2 PCR Step Activation Polymerase Activation HOLD HOLD CYCLE 2min 50 C 10 min 95 C 15 sec 95 C 1 min 60 C Thermal Cycling Conditions for One Step Sap Y instrument RESS T P C R m Thermal Cycler Protocol R Thermal Profile Auto Increment If you selected the one step RT PCR method cDNA m s TT m R generation and amplification take place simultaneously at this point in the workflow The following table shows the thermal cycling conditions for one step RT PCR experiments aia i ror Step Sample Volume 50 uL l Dissociation Protocol 60 0 E M 9600 Emulation Times and Temperatures One step RT PCR Initial Steps PCR Each of 40 Cycles Reverse Transcription AmpliTag Gold DNA Melt Anneal Extend Polymerase Activation HOLD HOLD CYCLE 30 min 48 C 10 min 95 C 15 sec 95 C 1 min 60 C Notes Relative Quantification Getting Started Guide for 7000 v1 1 25 Chapter 4 Generating Data from RQ Plates Specifying Thermal Cycling Conditions and Starting the Run To specify thermal cycling conditions and start the run
26. amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples e Quick spin PCR samples whenever residual sample is present on the inside lid such as after dropping a tube or when there is condensation on the tube from heating or thawing e Keep reactions and components capped as much as possible e Use aerosol resistant or positive displacement pipette tips e Clean lab benches and equipment periodically with freshly diluted 10 chlorine bleach Preparing the PCR Master Mix The second step in the two step RT PCR procedure is amplifying the cDNA TaqMan Universal Master Mix reagents provide a PCR mix that may be used with any appropriately designed primer and probe to detect any DNA or cDNA sequence The TaqMan Universal PCR Master Mix Protocol PN 4304449 explains how to use the reagents provided in the kit The following table lists the universal assay conditions volume and final concentration for using the master mix N or ene CHEMICAL HAZARD TaqMan Universal PCR Master Mix may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Reaction Component Mer eh 2 Final Concentration TaqMan Universal PCR Master Mix 2X 25 0 1X Forward primer 5 0 50 to 900 nM Reverse primer 5 0 50 to 900 nM TaqMa
27. an access the Tools menu In the Detector Manager select File gt New The New Detector dialog box opens In the New Detector dialog box enter a name for the detector IMPORTANT The name of the detector must be unique and should reflect the target locus of the assay such as GAPDH or RNase P Do not use the same name for multiple detectors Optionally click the Description field then enter a brief description of the detector Find Detector Name Description Reporter quencher Color Notes ile i Add To Plate Document Duplicate amp dd to Plate Document Import Export Clear Clear All Properties 2003 08 21 13 51 2003 08 21 13 51 2003 08 21 13 51 2003 06 21 13 51 FAM none FAM none Name GAPDH Description r Reporter Dye FAM FRANS Dye none x T Relative Quantification Getting Started Guide for 7000 v1 1 47 Appendix A 9 In the Reporter Dye and Quencher Dye drop down lists select the appropriate dyes for the detector Note The dyes that appear on the Reporter and Quencher Dye lists are those that have been previously entered using the Dye Manager If the dye that you want to use does not appear in a list use the Dye Manager to add the dye and then return to this step in this procedure Refer to the Online Help f
28. an overview Design an RQ experiment Reverse transcribe total RNA to cDNA PCR amplify the cDNA and collect data in RQ Plate documents Analyze one or more RQ Plates in an RQ study Introduction About the 7000 SDS Instrument Description The ABI PRISM 7000 Sequence Detection System 7000 SDS instrument is a second generation sequence detection instrument capable of quantitative and qualitative detection with fluorescent based PCR chemistries The instrument is capable of quantitative detection using real time analysis and qualitative detection using end point and dissociation curve analysis The 7000 SDS instrument combines thermal cycling fluorescence detection and application specific software It detects accumulated polymerase chain reaction PCR product cycle by cycle thus making quantification available immediately after completion of PCR without the need for further process analysis Supported Assay The 7000 SDS instrument allows you to perform the following assays with plates or Types tubes in the 96 well format e Relative Quantification RQ Determines the quantity of a single nucleic acid target sequence within an unknown sample relative to the same sequence within a calibrator sample e Absolute Quantification AQ Determines the absolute quantity of a single nucleic acid target sequence within a sample e Allelic Discrimination AD Indicates the genotype of samples e Plus Minus Indica
29. and Laboratory Use Part 1 General Requirements and EN 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials EMC This instrument meets European requirements for emission and immunity EMC Directive 89 336 EEC This instrument has been tested to and complies with standard EN 61326 Group 1 Class B Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment Relative Quantification Getting Started Guide for 7000 v1 1 Safety and Electromagnetic Compatibility EMC Standards Australian EMC Standards Relative Quantification Getting Started Guide for 7000 v1 1 XVII re you familiar with the 7000 instrument and relative quantification Have you designed your experiment Have you converted total RNA to CDNA Have you collected PCR data in an RQ plate document Have you analyzed data in an RQ study Chapter 1 Introduction Primer Oligo d T or random hexamer A strand Thermal Profile Auto Increment Stage 1 Stage 2 Rees Res Rep Chapte 4 assan A elative Quantification ontainer 96 well Clear Template fBlank Document Browse Read the introduction for
30. and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS e Handle chemical wastes in a fume hood e After emptying the waste container seal it with the cap provided e Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Relative Quantification Getting Started Guide for 7000 v1 1 Physical Hazard Safety Ultraviolet Light Physical Hazard Safety Ultraviolet Light ULTRAVIOLET LIGHT HAZARD Looking directly at a UV light source can cause serious eye damage Never look directly at a UV light source and always prevent others from UV exposure Follow the manufacturer s recommendations for appropriate protective eyewear and clothing Biological Hazard Safety General ANTENE BIOH
31. cation Getting Started Guide for 7000 v1 1 References Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 Livak K J and Schmittgen T D 2001 Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 4 T Method Methods 25 402 408 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Relative Quantification Getting Started Guide for 7000 v1 1 45 References 46 Relative Quantification Getting Started Guide for 7000 v1 1 Creating Detectors Before you can use a plate document to run a plate it must be configured with detector information for the experiment A detector is a virtual representation of a gene or allele specific nucleic acid probe reagent used for analyses performed on SDS instruments Examples of reagents represented as detectors include TaqMan probes and the SYBR Green 1 dsDNA binding dye You must create and apply detectors for all assays present on the plate before running it To create a detector 1 Notes Select Tools gt Detector Manager The Detector Manager dialog box opens Note A plate document any type must be open before you c
32. cription To view the plot Example Gene Expression Plot Orientation Detector value of a given sample Detectors are plotted on the x axis om i and each bar shows the detector 2 In the Orientation drop Jer piia E i l 1 In the RQ Results panel Dis pore Fa a cat select the Gene amaii o Y Gene Expression Expression tab down list select Detector Log 10 R FLT4 PDH Gene Expression Plot 1 In the RQ Results panel Orientation Sample select the Gene Samples are plotted on the x axis en a a and each bar shows the set of 2 In the Orientation drop HHH sample values of a given detector down list select Sample Notes 38 Relative Quantification Getting Started Guide for 7000 v1 1 Analyzing and Viewing the Results of the RQ Study Modifying Graph Settings Modifying Graph Settings You can modify the default graph settings for several graphs including Amplification and Gene Expression plots For example you can use labeled 3 D bars in the Gene Expression plot 1 Ina graph double click an axis of a plot or select aE ERE Fr HEFE e z Of ie neiinarinn iene Tx i Analysis gt Graphical Settings to display the ER Graph Settings dialog box Brd e To set real time settings change them before you start the run Auto Scale is the default Bar wd h 70
33. e Primer Express Software custom e PN 4330710 1 user license designed primers and probes PN 4330709 10 user license PN 4330708 50 user license Reagent tubes with caps 10 mL Applied Biosystems PN 4305932 Centrifuge with adapter for 96 well Major laboratory supplier MLS plates Gloves Major laboratory supplier MLS Microcentrifuge MLS Microcentrifuge tubes sterile 1 5 mL MLS Nuclease free water MLS Pipette tips with filter plugs MLS Pipettors positive displacement MLS Tris EDTA TE Buffer pH 8 0 MLS Vortexer MLS Notes 4 Relative Quantification Getting Started Guide for 7000 v1 1 Materials and Equipment Description of the Sample RQ Experiment Notes Relative Quantification Getting Started Guide for 7000 v1 1 5 Have you selected a PCR method Single or Multiplex page 7 Select a PCR method E Endo Ctrl T Target Have you designated the targets calibrator fa Designate the four endogenous control eae page9 essential components and replicates for the of Replicates of RQ experiments experiment Have you selected the SDS chemistry SYBR Green 1 or TaqMan and reagent configuration Select the SDS chemistry page 11 for your experiment Have you designed your primers and probes Choose primers and probes for your experiment Go to chapter 3 Performing Reverse Transcription Primer Extended on mRNA p Chapter
34. e the expression levels of several genes in the liver kidney and bladder tissue of an individual There are 23 genes of interest including ACVR1 ACVR2 CCR2 CD3D and FLT4 These genes are Once targets are identified you need to determine which samples will serve as the basis of comparison for the other samples in the study In this experiment the liver samples served as the calibrator The 7000 SDS instrument software sets gene expression levels for the calibrator samples to 1 Consequently if there is more ACRV1 in the kidney than in the liver the gene expression level of ACRV1 in the kidney is greater than 1 Similarly if there is less CD3D in the bladder than in the liver the gene expression level of CD3D in the bladder is less than 1 Because RQ is based on PCR the more template there is in a reaction the more the PCR product and the greater the fluorescence To account for possible differences in the amount of template added to the reaction GAPDH serves as an endogenous control Expression levels of the endogenous control are subtracted from expression levels of target genes The experiment includes three sets of endogenous controls one for each tissue Further the endogenous control for each tissue must be amplified on the same plate as the target sequences for that tissue Finally note that the experiment uses the singleplex PCR format and therefore the endogenous controls are amplified in different wells from the targets Fo
35. ect from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter gt View All Runs User Attention Two user attention words appear in Applied Biosystems user documentation Each word Words _ implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Relative Quantification Getting Started Guide for 7000 v1 1 Vil Preface Send Us Your Comments Safety Alert Words Related Documentation Examples of the user attention words appear below Note The size of the column affects the run time Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password IMPORTANT You must create a separate Sample Entry Spreadsheet for each 96 well microtiter plate Safety alert words also appear in user documentation For more information see Safety Alert Words on page x For more information about using the instrument and relative quantification refer to e ABI PRISM 7000 Sequence Detection System Online Help Livak K J and Schmittgen T D 2001 Analysis of Relative Gene Expression Data Us
36. ent a Add plates to the study b Configure analysis settings c Select samples for the study fs i Performing an RQ Study 2 View and analyze the results Creating an RQ Study Document To conduct a comparative analysis of RQ plates in a study you must first create anRQ Study document The 7000 v1 1 software with the RQ Study Add On uses the comparative method 2 of relative quantification For more information about methods of calculating relative quantification refer to ABI PRISM 7700 Sequence Detection System User Bulletin 2 In an RQ study you can You cannot e Select the endogenous control and the calibrator sample Create add or modify samples e Select the control type The 7000 SDS instrument e Create add or modify detectors software assigns the control type when applicable e Change detector tasks e Set baseline and threshold parameters and RQ Min Max Confidence Levels e Omit individual wells or sample replicates Notes Relative Quantification Getting Started Guide for 7000 v1 1 33 Chapter 5 Performing an RQ Study Creating an RQ Study Document To create a new RQ Study document 1 Select File gt New The New Document dialog box opens New Documen t Container SEE Clear 2 Inthe Assay drop down list select Relative Quantification ddCt Study Accept the default Template Blank Document settings for the Container and Tem
37. er Liver Liver Liver ff A ff D Liver Liver Live Live Livi Live Lei Lei Live Sample Name E iver Liver Detector Task and Liver LIVET Ler LIVET Cher LIVET Iver EH mM i S GE F Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver E E E E G Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver T H Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver 3 m m m a G For a sample illustration of how a multiplexed plate would appear refer to the Comparative Method topic of the Online Help for the 7000 SDS instrument Notes 24 Relative Quantification Getting Started Guide for 7000 v1 1 Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Cycling Conditions for PCR Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Cycling Conditions for Sete Ynstvarnent Wren PCR Thermal Cycler Protocol Thermal Profile Auto Increment If you selected the two step RT PCR method for your me Res Pee RQ experiment recommended you have already completed the RT step At this point in the workflow you are ready to PCR amplify cDNA The default thermal cycling conditions for the PCR step of the procedure shown in the following table Ada cyete Add Hold Add step Sample Vole Fay ta should appear on the Instrument t
38. es and delivers quality controlled primer and probe sets Use this service if the assay you need is not currently available To place an order contact your Applied Biosystems representative Primer Express Software Helps you design primers and probes for your own quantification assays For more information about using this software refer to the Primer Express Software v2 0 User s Manual PN 4329500 Applied Biosystems provides Assay Design Guidelines which have been developed specifically for quantification assays When used in their entirety these steps provide a rapid and reliable system for assay design and optimization For information about the Assay Design Guidelines refer to the Sequence Detection Systems Chemistry Guide Premade assays from Assays on Demand products are available for all the genes included in the sample experiment primers and probes were obtained from Applied Biosystems Each assay consists of two unlabeled PCR primers forward and reverse and a FAM dye labeled TagMan MGB probe provided as a 20X assay mix If you order Assays on Demand or Assays by Design products probes are already labeled with a reporter dye If you design your own assays you would need to specify a reporter dye for your custom probe s For singleplex experiments where the targets and endogenous controls are amplified in separate wells you can use the same dye for targets and endogenous control s For multiplex experimen
39. es the instrument has e Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Read and understood all applicable Material Safety Data Sheets MSDSs See About MSDSs on page xiii Nem PHYSICAL INJURY HAZARD Use this instrument as specified by Applied Biosystems Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Relative Quantification Getting Started Guide for 7000 v1 1 Chemical Safety NS Chemical Hazard Warning Chemical Safety Chemical Hazard Nem CHEMICAL HAZARD Before handling any chemicals refer to Warning the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining You can obtain from Applied Biosystems the MSDS for any chemical supplied by MSDSs Applied Biosystems This service is free and available 24 hours a day To obta
40. ettings Relative Quantification a Select Analysis gt Analysis Settings The ke i Detector all Analysis Settings dialog box opens Auto Ct b In the Detectors drop down list select All C Manual Ct Threshold a 200000 c Select AutoCt or Manual Ct es e Ifyou select AutoCt the SDS software C Marul Baseline Start cycle Auto End cycle Auto automatically calculates both the eee threshold and baseline After analysis F EEI you must verify that the baseline and O EO threshold were called correctly for i Control Type Multplezed Non multiplexed each detector Refer to the Online Help for the 7000 SDS instrument RO Min Max Confidence 9500 x e Ifyou select ManualCt specify both the threshold and the baseline for the FEER ae selected detector s d Select the calibrator sample Note As discussed in Specifying the Components of an RQ Experiment on page 9 if your experiment uses only a single plate there must be at least two different samples that have different names and have their own endogenous controls You can go back to a saved RQ Plate document and change the sample names if necessary e Select the Endogenous Control Detector f Select the Control Type if the study contains peepee coarse EE both multiplex and nonmultiplex reactions Fi SST ai ene Expres ee ee a ffi Aug 05 2003 02 05 PM Au a 05 2003 02 05 PM Note Ifthe
41. g viii 2 template documents 24 text conventions vii thermal cycling conditions default for PCR 25 one step RT PCR 25 specifying 26 thermal cycling parameters for plates added to a study 34 RT using High Capacity cDNA Archive kit 16 threshold cycle definition 27 setting for RQ studies 35 Training obtaining information about viii training obtaining information about 2 types of assays 1 U ultraviolet light xv uracil N glycosylase 12 US safety standards xvi V VIC dye 13 W WARNING description x Well Information tab 36 Well Inspector dialog box 23 wells replicate 9 54 Relative Quantification Getting Started Guide for 7000 v1 1 Services and Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Services and Support At the Services and Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Services and Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 To
42. geureux Lire les fiches techniques de s ret de mat riels avant la manipulation des produits CAUTION Hot surface ATTENTION Surface br lante DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils L instrument ne contient aucune piece r parable par l utilisateur Toute intervention doit tre effectu e par le personnel de service qualifi de Applied Biosystems ATTENTION Parties mobiles CAUTION Moving parts General Instrument Safety Moving and Lifting the Instrument Operating the Instrument xii N 70 PHYSICAL INJURY HAZARD The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Ensure that anyone who operat
43. hat contain PCR reagents for the amplification of target sequences The instrument software indicates targets by a jj e Endogenous Control All detectors of wells that contain reagents for the amplification of the endogenous control sequence The instrument software indicates endogenous controls by an J Notes 22 Relative Quantification Getting Started Guide for 7000 v1 1 To create a new plate document 1 6 Select Start gt Programs gt ABI Prism 7000 gt ABI Prism 7000 SDS Software to start the 7000 SDS instrument software Select File gt New The New Document dialog box opens In the Assay drop down list select Relative Quantification ddCt Plate Accept the default settings for the Container and Template fields 96 Well Clear and Blank Document Click OK The 7000 SDS instrument software opens a new RQ Plate document Specify the detectors for the plate a Select Tools gt Detector Manager The Detector Manager dialog box lists all detectors that have been created for the system If no detectors are listed in the Detector Manager dialog box create detectors as explained in Appendix A Creating Detectors b Click on the detector name s to select the appropriate detector s c Click Add to Plate Document The detectors are added to the plate document d Click Done or xj to close the Detector Manager Note After adding detectors to a document the Detector Manage
44. ilable for Configurations for quantitative experiments The reagent configuration you use depends on whether you are RT PCR performing one step or two step RT PCR In one step RT PCR reverse transcription RT and PCR are take place in a single buffer system that is a single tube or well This offers the convenience of a single tube preparation for RT and PCR amplification However the carryover prevention enzyme AmpErase UNG uracil N glycosylase cannot be used with one step RT PCR For more information about UNG refer to the Sequence Detection Systems Chemistry Guide e Two step RT PCR is performed in two separate reactions total RNA is reverse transcribed into cDNA which is then amplified by PCR This method is useful for detecting multiple transcripts from a single cDNA template or for storing cDNA aliquots for later use If dUTP is not used during the RT step AmpErase UNG enzyme can be used to prevent carryover contamination IMPORTANT Applied Biosystems recommends that you use the two step RT PCR method for RQ experiments This guide assumes that RQ experiments are designed using two step RT PCR Further only the recommended reagent configurations are documented For additional options refer to the Sequence Detection Systems Chemistry Guide The following table lists the recommended kits for two step RT PCR For information about the available reagents for one step RT PCR refer to the Sequence Detection Systems Chemis
45. in MSDSs 1 Go to https docs appliedbiosystems com msdssearch html 2 Inthe Search field type in the chemical name part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose 4 To have a copy of a document sent by fax or e mail select Fax or Email to the left of the document title in the Search Results page then click RETRIEVE DOCUMENTS at the end of the document list 9 After you enter the required information click View Deliver Selected Documents Now Chemical Safety To minimize the hazards of chemicals SuIgeIInes Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page xiii e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Relative Quantification Getting Started Guide for 7000 v1 1 xili Safety and EMC Compliance Information Chemical Waste Safety e Minimize the inhalati
46. ing Real Time Quantitative PCR and the 2 Method Methods 25 402 408 e Sequence Detection Systems Chemistry Guide Chemistry Guide PN 4330019 ABI PrisM 7700 Sequence Detection System User Bulletin 2 Relative Quantitation of Gene Expression PN 4303859 Send Us Your Comments viii Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com Relative Quantification Getting Started Guide for 7000 v1 1 Safety and EMC Compliance Information This section includes the following topics gt Safety Conventions Used in This Document 20sseeeeeeeeeee X gt Symbols on Instruments cee eens Xi Safety Labels on Instruments eee ens xii General Instrument Safety skr k Er edett hous RARE ERE xii gt Chemical Safe au wegeces grtt up t tS Erre EEEREN xiii Chemical Waste Safety 0 ee eee eee eeees XIV Physical Hazard Safely iidacitanseredes onedebu oe ue 246 dea seat acca XV Biological Hazard Safety sisscasvcviseusd steeewewaaw dad seen nee ons XV Workstation Safety esser REE eee TERRE ENEN ERa XV Safety and Electromagnetic Compatibility EMC Standards XV Relative Quantification Getting Started Guide for 7000 v1 1 Ix Safety and EMC Compliance Information Safety Conventions Used in This Document Safety Conventions Used in This Document Safety Alert Words
47. instrument records the fluorescent emissions resulting from cleavage of TaqMan probes in the presence of the target and reference sequences All data generated during the run are saved to the RQ Plate document that you specified in step 3 Notes 26 Relative Quantification Getting Started Guide for 7000 v1 1 Analyzing and Viewing RQ Plate Data Terms Used in Quantification Analysis Analyzing and Viewing RQ Plate Data Terms Used in Following are terms commonly used in quantification analysis Quantification Analysis Term Definition Baseline A line fit to the initial cycles of PCR in which there is little change in fluorescence signal For information about setting the baseline refer to the Online Help for the 7000 SDS instrument Threshold cycle Cy The fractional cycle number at which the fluorescence passes the threshold For information about setting the threshold refer to the Online Help for the 7000 SDS instrument Passive reference A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescent fluctuations caused by changes in concentration or of volume Reporter dye The dye attached to the 5 end of a TaqMan probe The dye provides a signal that is an indicator of specific amplification Normalized reporter The ratio of the fluorescence emissio
48. is a multi plate document called the RQ Study You can analyze up to ten RQ plates in a study RQ Study documents neither control the instrument nor do they provide tools for setting up or modifying plates The following figure illustrates the RQ Study process Kidney _ bres EES bladderplate sds Plated Reactions 7000 SDS Instrument v1 1 Software RQ Plate Documents v1 1 Software RQ Study Document with RQ Study with RQ Study d On Add On IMPORTANT The 7000 SDS instrument software v1 1 with the RQ Study Add On uses only the comparative method AACt to calculate relative quantities of a nucleic acid sequence About the Sample RQ Experiment Description of the This guide uses a sample RQ experiment to help you understand the workflow Sample RQ Experiment In the sample RQ experiment levels of expression of 23 genes were compared in the liver kidney and bladder tissue of an individual The experiment was designed for singleplex PCR samples and endogenous controls were amplified in separate wells Glyceraldehyde 3 phosphate GAPDH served as the endogenous control Four replicates of each sample and endogenous control were amplified In this experiment an entire 96 well plate is devoted to each tissue because the four replicates of each of the 23 genes plus the endogenous control take up all 96 wells Predesigned and labeled primer probe sets were selected from the Applied Biosystems As
49. ll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com Applied Bibsystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in USA 09 2003 Part Number 4346727 Rev A an Applera business
50. mary csv e Well Information csv e Both csv 2 Enter a file name for the export file Note The name of the dialog box depends on the type of data you want to export 3 Click Save Notes Exporting RQ Study Results is ABI Prism 7000 SDS Software Plate1 Relative Qt f File View Tools Instrument New Ctrl M Analysis fel Omit Pata rt ys Well Position Open Ctrl O ee if SE Mis 7 Flate Y Amplification E Close Save Ctrl 5 eshold Auto CT Save 45 regl 1692 1890 auc 42 Auto Import Sample Setup Export Sample Setup 40 Calibration Data Fage Setup Print Preview Spectra Print Ctrl F Component Delta Rn 1 Liver sds ae Exit __ MHs A o Sample Summary ER RC AR 0 209008 Well Information NFA 0 174955 Auto Se 0 350872 Auto 30 PTGFR d elect Results Export File Save in SDS Documents gt e fr Fee SampleComponenExportFile cs SampleSpectraExportFile csy History i Desktop My Documents My Computer was se My Network P Cancel LZ SampleResultsE xportF ile Results Export File x File name Save as type Relative Quantification Getting Started Guide for 7000 v1 1 43 LER Chapter 5 Performing an RQ Study Exporting RQ Study Results Notes 44 Relative Quantifi
51. n intensity of the reporter dye Rn to the fluorescence emission intensity of the passive reference dye Delta R AR The magnitude of the signal generated by the given set of PCR conditions AR R baseline The figure below shows a representative amplification plot and includes some of the terms defined above Threshold No Template Control Cx 0 5 10 15 20 25 30 35 40 Cycle Number Starting the To analyze RQ Plate data after the run select Analysis gt Analyze The SDS software Analysis mathematically transforms the raw fluorescence data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dyes Based on that comparison the software generates several types of result views as described in the following section Notes Relative Quantification Getting Started Guide for 7000 v1 1 27 Chapter 4 Generating Data from RQ Plates Analyzing and Viewing RQ Plate Data Types of Result Viewing the analysis results helps you verify that the cDNA was correctly amplified Views The four types of result views are shown in the following table View Description To view the plot Example Plate In the Results tab click the Plate Displays the sample name SUD FAD detector task and color and The plate appears on the screen R value Sample Name Rn value Detector Task and Color Spect
52. n probe 5 0 50 to 250 nM CDNA sample 5 0 10 to 100 ng Nuclease free water 5 0 Total 50 0 Notes 20 Relative Quantification Getting Started Guide for 7000 v1 1 Preparing the PCR Master Mix Preventing Contamination For most TaqMan reagent or kit based assays that are designed and run following Applied Biosystems assay development guidelines refer to the Sequence Detection Systems Chemistry Guide using a concentration of 900 nM primers and a 250 nM probe provides a highly reproducible and sensitive assay when using cDNA or DNA as a substrate in a singleplex assay Probes and primers that you design using Primer Express software must be optimized to work with the universal assay conditions using the volumes listed in the table on page 20 All Assays by Design ABD and Assays on Demand AOD products are formulated so that the final concentration of the primers and probes falls within the recommended parameters But because the primers and probes are supplied as a 20X assay mix the volumes are slightly different from the universal assay conditions as explained in the following Example NT CHEMICAL HAZARD TaqMan Universal PCR Master Mix may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Primers and probes for the sample RQ e
53. ne expression levels of the following genes when the liver tissue was used as the calibrator ACVR1 ACVR2 CCR2 CD3D and FLT4 Selecting the detectors in the RQ Detector grid 1 displays the sample information in the RQ Sample grid 2 and a result graph in the RQ Results panel 3 Note that e The Gene Expression tab is selected e The gene expression levels are sorted by detector e Gene expression levels for bladder samples are denoted by the green bar those for kidney samples by the blue bar These colors are used to denote the samples in the RQ Sample Grid and the RQ Results Panel plots e Liver samples were used as calibrators consequently the expression level of these samples is set to 1 But because the graph plots gene expression levels as log values and the log of 1 is 0 the expression level of the calibrator sample appears as 0 in the graph e Because the relative quantities of the targets are normalized against the relative quantities of the endogenous control the expression level of the endogenous control is 0 there are no bars in for GAPDH e The value in the RQ column in the Sample Summary tab is 24407 73 ABI Prism 7000 SDS Ease A Fie View Tools trume ent Analysis el amp a E Find 0 421692 Auto Auto 40 Auto Auto mua B We 0 20 l 0 40 0 60 10 x T PER y Help e xi E E We E Omit Data Rn vs Cycle i SIN x Calibrator 7 Plate Y Amplificati
54. on Plot x Orientation Detector Gene Expression 0 20 GTF2B 0 284137 GTF2I 0 320520 0 000 1 000 0 8 3 067 0 119 0 1 40 0 005 1 003 0 0 000 1 000 Log 10 Relative Quantification ACYR1 ACYR2 CCR2 CD3D FLT4 GAPDH X Sample Summary A Well Information 7 Ready Disconnected ESES SSS SZ Detectors Notes 36 Relative Quantification Getting Started Guide for 7000 v1 1 Analyzing and Viewing the Results of the RQ Study Viewing Amplification Plots Analyzing and Viewing the Results of the RQ Study Viewing Amplification Plots show either of the following Amplification p Plots e The fluorescence of each detector as a function of cycle number e The threshold cycle C as a function of well position View Description To view the plot Example Rn vs Cycle Linear 1 In the RQ Results panel select the Amplification ce ERE The Rn vs Cycle plot displays Plot tab normalized reporter R dye fluorescence as a function of cycle 2 In the Data drop down list You can use this plot to identify and select Rn vs Cycle examine irregular amplification For more information about R refer to the online help for the 7000 SDS instrument ARn vs Cycle Log 1 In the RQ Results panel I select the Amplification 7 Plate Y Amplification Plot Y 4 The AR i e ARn vs Cycle plot displays Rn o ab
55. on of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS e Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical Waste Safety Chemical Waste NT HAZARDOUS WASTE Refer to Material Safety Data Sheets and Hazard local regulations for handling and disposal ANITY CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death AINTE CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical Waste To minimize the hazards of chemical waste Safety Guidelines XIV Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste e Provide primary
56. or the 7000 SDS instrument for more information 6 Click the Color box select a color to represent the detector using the Color dialog box then click OK 7 Optionally click the Notes field then enter any additional comments for the detector 8 Click OK to save the detector and return to the Detector Manager dialog box 9 Repeat steps 2 through 8 for the remaining detectors 10 In the Detector Manager dialog box click Done when you have finished adding detectors g In the sample RQ experiment a detector was created for each target gene and endogenous control 24 detectors were created 23 for the target genes and 1 for the endogenous control GAPDH For example the detector for the ACVR1 gene was named ACVR1 and assigned a yellow color Because all Assays on Demand products have probes that are labeled with FAM dye FAM was selected for the reporter dye Additionally Assays on Demand products use TaqMan MGB probes which do not need quenchers no quencher dye was selected for the detector Assays on Demand products are shipped with an assay information file AIF This text based file contains information about the assays that you ordered including the Applied Biosystems Assay ID number well location of each assay primer concentration and primer sequence The file also indicates the reporter dyes and quenchers if applicable that are used for each assay When creating detectors you would use the reporter
57. p Type Time Temperature HOLD 10 min 25 C HOLD 120 min 37 C Note Thermal cycling conditions for one step RT PCR are described in Thermal Cycling Conditions for One Step RT PCR on page 25 16 Relative Quantification Getting Started Guide for 7000 v1 1 Generating CDNA Storing CDNA Storing CDNA After thermal cycling store all cDNA samples at 15 to 25 C To minimize repeated freeze thaw cycles of cDNA Applied Biosystems recommends that you store your cDNA samples in aliquots Nore CHEMICAL HAZARD 10 x RT Buffer may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves For the sample experiment RNA was extracted from the liver bladder and kidney tissues of an individual RNA concentration was determined through spectrophotometry using A260 and the RNA was diluted to a final concentration of 50 ng uL The RT master mix was prepared as follows using guidelines from the High Capacity cDNA Archive Kit Protocol Component Volume uL Reaction 10X Reverse Transcription Buffer 10 25X dNTPs 4 10X random primers 10 MultiScribe Reverse Transcriptase 50 U uL 5 Nuclease free water 21 Total per reaction 50 The cDNA archive plate was then prepared by pipetting e 50 uL of the RT master mix e 30 uL of nuclease free water e 20 uL of
58. plate fields Browse 96 Well Clear and Blank Document OK Cancel 3 Click OK The SDS software opens a new RQ Study document and displays the RQ Study Main View The RQ Study Main View has three frames a ar a a a a a RQ Detector Grid Allows you to select z HE Pr aac Yo OO E detectors to associate with the loaded study The following information is displayed for each detector Color Detector name Threshold Value Auto Ct and Baseline b RQ Sample Grid Displays the samples associated with the selected detector s The Sample Grid displays numerical results of RQ computations and has two subtabs Sample Summary or Well Information c RQ Results Panel Contains the three eee L asda results based tabs Plate Amplification Plot and Gene Expression Nm ZA 4 Add plates to the study a Click Add Plate The Select RQ Plate s i mes E l O e uo dialog box opens E i zx b Select the plate s that you want to add to the study then click Open The selected dd plates are displayed in the Plate tab K IMPORTANT All plates added to a study nl must have identical thermal cycling parameters the same number of steps eS cycles sample volume emulation mode The SDS software will reject a plate if it detects any differences Notes 34 Relative Quantification Getting Started Guide for 7000 v1 1 5 n Configure analysis settings Analysis S
59. quate accessibility to the keyboard monitor and mouse e Position the keyboard mouse and monitor to promote relaxed body and head postures Relative Quantification Getting Started Guide for 7000 v1 1 XV N Safety and EMC Compliance Information Safety and Electromagnetic Compatibility EMC Standards Safety and Electromagnetic Compatibility EMC Standards U S and Canadian Safety Standards c UL us Canadian EMC Standard European Safety and EMC Standards CE Australian EMC Standards XVI This section provides information on U S and Canadian Safety Standards e Canadian EMC Standard European Safety and EMC Standards Australian EMC Standards This instrument has been tested to and complies with standard UL 3101 1 Safety Requirements for Electrical Equipment for Laboratory Use Part 1 General Requirements This instrument has been tested to and complies with standard CSA 1010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements This instrument has been tested to and complies with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Safety This instrument meets European requirements for safety Low Voltage Directive 73 23 EEC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control
60. r Bladder i 3ladder d Li Notes 40 Relative Quantification Getting Started Guide for 7000 v1 1 Omitting Samples from a Study Omitting Samples from a Study For any PCR experimental error may cause some wells to amplify insufficiently or not at all These wells typically produce C values that differ significantly from the average for the associated replicate wells If included in the calculations these outliers can result in erroneous measurements To ensure precise relative quantification you must carefully view replicate groups for outlying wells You can remove outliers manually using the C vs Well Position Amplification Plot To remove samples from an RQ Study Ctvs Well Position Amplification Plot 1 Select the Amplification Plot tab 2 Inthe Data drop down list select Ct vs Well Position 3 Inthe RQ Detector grid select a detector to examine All samples that use this detector are displayed in the RQ Samples grid 4 Inthe RQ Samples grid click to select the samples to display in the Amplification Plot 5 Verify the uniformity of each replicate population by comparing the groupings of C values for the wells that make up the set Plate Y Amplification Plot Gene Expression ACYRI 0 421692 Auto ACVR2 10 401890 Auto CCR2 0 055709 Auto 0 307250 10 200000 fm 0 352249 GAPDH 0415943 Auto G
61. r remains open until you close the window Label the wells of the plate a Select View gt Well Inspector The Well Inspector dialog box lists the detectors that you added in step 5 b Click a well to select it If there are replicate wells for a sample click on all the wells to select them c In the Well Inspector enter the sample name Notes Creating an RQ Plate Document Detector Tasks AZIAN Container FAM none FAM none 2 3 2003 08 21 13 2003 08 21 13 2003 08 21 13 2003 08 21 13 2003 08 21 13 2003 08 21 14 1 2003 08 21 14 1 2003 08 21 14 1 VIC FAM none FAM none FAM none FAM FAM fane IMN3M874 wal O ta ee File v Add To Plate Document Done 96 Well Clear Z Template Blank Document Y Browse OK Cancel Detector Manager x i m Detector List Find a Detector Hame ACYR1 FAM none 2 ACYR2 FAM none Selected detectors Relative Quantification Getting Started Guide for 7000 v1 1 23 Chapter 4 Generating Data from RQ Plates Creating an RQ Plate Document 7 In the Well Inspector select the detector s and detector tasks for each sample a Click on a detector to select it b Click the Use check box c Click under the Task column to assign the Detter Reporter Yuenchor lask Color detector task FE PEER CORE SE d Repeat
62. r than 1 9 e It should be intact when visualized by gel electrophoresis It should not contain RT or PCR inhibitors The High Capacity cDNA Archive Kit Protocol 4312169 contains additional guidelines for preparing the RNA template The High Capacity cDNA Archive Kit 1s optimized to convert 0 1 to 10 ug of total RNA to cDNA Convert enough total RNA so that the final concentration of total RNA converted to cDNA is 10 to 100 ng in 5 UL for each 50 uL PCR reaction Generating cDNA Using the High Capacity cDNA Archive Kit Thermal Cycling Parameters for RT Notes As mentioned in About Reagent Configurations for RT PCR on page 12 Applied Biosystems recommends that you use the two step RT PCR method for RQ experiments Synthesis of cDNA from total RNA samples using the High Capacity cDNA Archive Kit PN 4322171 is the first step in the two step RT PCR procedure Use the manual method for converting total RNA into cDNA as specified in the High Capacity cDNA Archive Kit Protocol PN 4322169 Note The protocol is not shipped with the High Capacity cDNA Archive Kit You must download the protocol from http docs appliedbiosystems com search taf To search for the document select ABI PRISM 6100 Nucleic Acid PrepStation under Products then click Search The protocol is listed under the Protocols heading The High Capacity cDNA Archive Kit uses the following thermal cycling parameters for the RT step Ste
63. ra 1 In the Results tab click the Spectra subtab Displays a plot of spectra Ea raw fluorescence data for The Cycle Number slider is at 1 the selected cycle number and the plot is empty until you and well s select wells The plot varies depending on 2 Click a well to include it in the the reporter dye used in the plot Ctrl click to include multiple assay The example on the wells Click drag to include right shows a successful multiple adjacent wells emp Ge DR Wen ein 3 Move the slider to indicate the WH o o reporter dye is used cycle number Component 1 In the Results tab click the Displays a plot of Component subtab fluorescence level of each The graph is blank until you dye against the cycle number select wells for selected well s 2 Click on a well to include it in the plot Ctrl click to include multiple wells Click drag to include multiple adjacent wells Mr Fan MV Fox Notes 28 Relative Quantification Getting Started Guide for 7000 v1 1 Analyzing and Viewing RQ Plate Data Reanalyzing Data View Description To view the plot Example Amplification Plot This view displays a plot of R against cycle number for the selected detector and well s 1 In the Results tab click the Amplification Plot subtab The plot is empty until you select a detector or all detectors and well s 2 Click
64. s contacting viii 2 customer feedback on documentation viii Services and Support viii 2 Technical Communications viii Technical Support viii 2 Assay Design Guidelines 13 21 assay information files 48 assay types Assays by Design 13 Assays on Demand 13 Australian EMC standards xvi B baseline 27 biological hazard safety xv C calibrating the 7000 SDS instrument 19 calibrator definition 9 selecting in RQ studies 35 Canadian safety standards xvi CAUTION description x Relative Quantification Getting Started Guide for 7000 v1 1 Index cDNA generating 16 storing 17 chemical safety guidelines xiii comparative method of calculation 3 33 Component view 28 confidence levels 35 contamination preventing 19 conventions text vii Ct vs Well Position view 37 Ct See threshold cycle curves standard 2 D DANGER description x data analyzing 27 37 exporting 30 43 generating PCR data from RQ plates 26 omitting from a study 41 delta Rn 27 Delta Rn vs Cycle view 37 designing RQ experiments determining reagent configuration 11 PCR method 7 primers and probes 13 selecting an SDS chemistry 11 workflow 7 Detector Manager dialog box 23 47 detector tasks definition 22 specifying for RQ plates 24 detectors adding to RQ plates 23 creating 47 definition 47 selecting for RQ studies 35 documentation feedback viii documents exporting 30 43 importing 24 RQ Plate 22 51 Index documents con
65. s to a step in the analysis process Setup Instrument and Results The Results tab has several subtabs for the various viewers associated with an assay Run Setup Before creating an RQ Plate document you must define several parameters for each RQ Requirements plate e Detectors Determine which detector you will use for each sample and endogenous control Appendix A Creating Detectors explains how to create detectors IMPORTANT To conduct a comparative analysis of the data in a study all the plates in the study must contain a common set of detectors e Endogenous control s If your experiment consists of multiple plates each plate must have at least one endogenous control with at least three replicates If your experiment consists of a single plate with multiple samples there must be an endogenous control for each sample Specifying the Components of an RQ Experiment on page 9 explains the concept of an endogenous control All plates must use the same endogenous control for example GAPDH IMPORTANT In order to open RQ Plate or RQ Study documents you must have the RQ Study Software installed as explained in Appendix B Detector Tasks A task is a setting that you apply to the detectors within a well of a plate document and that determines the way the software uses the data collected from the well during analysis For RQ Plate documents there are two types of tasks Target All detectors of wells t
66. says on Demand product line Reactions were set up for two step RT PCR where the High Capacity cDNA Archive Kit and the TaqMan Universal PCR Master Mix were used for reverse transcription and PCR respectively Data was generated by running three RQ plates one for each tissue All three plates were analyzed in an RQ study with the Liver samples serving as the calibrator The implementation details of this sample RQ experiment will be discussed throughout this guide in Example boxes like this one Notes Relative Quantification Getting Started Guide for 7000 v1 1 3 Chapter 1 Introduction Materials and Equioment Materials and Equipment You need to supply the following items to complete an RQ study Item Source High Capacity CDNA Archive Kit Applied Biosystems PN 4322171 TaqMan Universal PCR Master Mix Applied Biosystems PN 43804437 MicroAmp Optical 96 Well Reaction Applied Biosystems Plate and Optical Caps PN 403012 Note Reaction plates and caps may also be purchased separately Reaction plates can also be sealed with ABI PRISM Optical Adhesive Covers Labeled primers and probes from one of the following sources e Assays on Demand Gene e Applied Biosystems Web site Expression Products predesigned primers and probes e Contact your Applied Biosystems e Assays by Design service Sales Representative predesigned primers and probes
67. steps a to c until all detectors and detector tasks have been specified 8 Accept the default setting for the Passive Reference ROX dye 9 Click xj to close the Well Inspector 10 Verify the information on each well in the Setup tab Note You can import sample information from spreadsheets or use template documents to set up plate documents Refer to the Online Help for the 7000 SDS instrument for more information about these tasks In the sample RQ experiment the samples for each of the three tissues liver kidney and bladder were plated on three separate plates Consequently there were three RQ Plate documents created one for each of the sample plates Because it is a singleplex experiment there is only one sample either a target or endogenous control but not both in each well Each well is associated with a detector indicated by the colored squares Additionally each well has also been assigned a detector task T target or E endogenous control The figure below shows a sample RQ Plate document after sample names detectors and detector tasks have been assigned for each well in the liver plate EE ee ee ee ee ee ee ee ee eee A Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver B Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver Liver 7 A fF fT FF E E C Liver Liver Liver Liver Liver Liver Liv
68. study contains only singleplex g 2 a EE reactions Nonmultiplexed is automatically selected if it contains only multiplex reactions Multiplexed is automatically selected Only when a study contains both types of reactions are you asked to indicate the control type g Select the RQ Min Max Confidence level em arne A HER n KRITIENA h Click Ok amp Aesnayze The detector information appears in the RQ Detector grid For more information about the settings in the Analysis Settings dialog box refer to the online help for the 7000 SDS instrument Notes Relative Quantification Getting Started Guide for 7000 v1 1 35 Chapter 5 Performing an RQ Study Creating an RQ Study Document 6 In the RQ Detector Grid select detectors to include in the result graphs by clicking a detector Ctrl click to include multiple detectors Click drag to include multiple adjacent i detectors pLa 1038720 GTF2B 0 284137 GTF2l 0 320520 X Orientation Auto The corresponding samples appear in the RQ Sample Grid Depending on which tab you select in the RQ Results Panel Plate Amplification Plot or Gene Expression analysis results are displayed Detectors To see information about a specific well select the Well Information tab Disconnected Suppose that you wanted to view the comparative ge
69. t RQ experiments require A target The nucleic acid sequence that you are studying A calibrator The sample used as the basis for comparative results e An endogenous control A gene present at a consistent expression level in all experimental samples By using an endogenous control as an active reference you can normalize quantification of a cDNA target for differences in the amount of cDNA added to each reaction Note that Each sample type for example each tissue in a study comparing multiple tissues requires an endogenous control Ifsamples are spread across multiple plates each plate must have an endogenous control Typically housekeeping genes such as B actin glyceraldehyde 3 phosphate GAPDH and ribosomal RNA rRNA are used as endogenous controls e Replicate wells For relative quantification studies Applied Biosystems recommends the use of three or more replicate reactions per sample and endogenous control to ensure statistical significance Replicates allow you to preserve data and remove outliers For more information about these concepts refer to the Sequence Detection Systems Chemistry Guide Notes Relative Quantification Getting Started Guide for 7000 v1 1 9 Chapter 2 Designing an RQ Experiment Specifying the Components of an RQ Experiment the targets An endogenous control was prepared for each tissue The objective of the sample experiment is to compar
70. temperature Nem CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves JN uld ELECTRICAL HAZARD Failure to ground the instrument properly can lead to an electrical shock Ground the instrument according to the provided instructions Relative Quantification Getting Started Guide for 7000 v1 1 Symbols on Instruments Electrical Symbols on Instruments Symbols on Instruments Electrical The following table describes the electrical symbols that may be displayed on Symbols on Applied Biosystems instruments Instruments Symbol Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates the On Off position of a push push main power switch Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a terminal that can receive or supply alternating current or voltage u eoreo Indicates a terminal that can receive or supply alternating or direct current or voltage Safety Symbols The follo
71. tes the presence or absence of a specific target sequence in a sample For more information about the assay types refer to the Sequence Detection Systems Chemistry Guide and the Online Help for the 7000 SDS instrument About Relative Quantification Real time PCR Real time PCR is the ability to monitor the progress of the PCR as it occurs Data is Assays collected throughout the PCR process rather than at the end of the PCR process end point PCR In real time PCR reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated at the end of PCR There are two types of quantitative real time PCR absolute and relative Notes Relative Quantification Getting Started Guide for 7000 v1 1 1 Definition of Relative Quantification Chapter 1 Introduction About RQ Experiments Relative quantification describes the change in expression of the target gene in a test sample relative to a calibrator sample The calibrator sample can be an untreated control or a sample at time zero in a time course study Livak and Schmittgen 2001 For example relative quantification is commonly used to compare expression levels of wild type versus mutated alleles or the expression levels of a gene in different tissues Unlike absolute quantification relative quantification provides accurate comparison between the initial level of template in each sample
72. the Components of an RQ Experiment 0 0000 cee eee 9 Selecting the Sequence Detection Chemistry and Reagent Configuration 11 Choosing the Probes and Primers 0 000 eee eee ee ees 13 Chapter 3 Performing Reverse Transcription 15 WVOVMMOW a reaa Eea le ab aA garam E a a deere ede Ae eae ai AS 15 REPORMENOdS st tse ler ere ALE bd coe aise ME enon bee ante ar bias od 15 Guidelines for Preparing RNA 0 0 cee eee eens 16 Generating CDNA v2 25st eae aes eee ad ie a Me ee a ee 16 Chapter 4 Generating Data from RQ Plates 19 VOTO aie steele Sencar oe Aart Sieh eM Daan Oa eee ek E N cae ecb tent 19 Before YOU BegIN toe veh ae ee NA Ga ea ed Re wo 19 Preparing the PCR Master Mix eee eee een eet ee eee eee 20 Creating an RQ Plate Document 0000 cc ee eee es 22 Specifying Thermal Cycling Conditions and Starting the Run 25 Analyzing and Viewing RQ Plate Data 0 0 ees 27 Exporting RQ Plate Documents 0000 cee et eee eee 30 IV Relative Quantification Getting Started Guide for 7000 v1 1 Chapter5 Performing an RQ Study 33 WOrKIOW cerida seo nt tp Soci bape whe Ed ser sedlen bard end ay duc ve ate Re at saa es ead eee ah aut O 33 Creating an RQ Study Document 0 0 0 cc eee eee 33 Analyzing and Viewing the Results of the RQ Study 000 02 e ce eee 37 Reanalyzing an RQ Study nadaan aore eee 40 Omitting Samples from a Study
73. tinued RQ Study 33 templates 24 dyes FAM 13 multiple 7 reporter 27 ROX 24 SYBR Green I 11 12 47 VIC 13 E electromagnetic compatibility standards See EMC standards EMC standards xvi Australian xvi Canadian xvi European xvi emulation mode 9600 26 endogenous controls associating with detectors 22 24 definition 9 for RQ plates 22 selecting for RQ studies 35 endpoint PCR 1 equipment 4 ergonomic safety xv European safety standards xvi exporting data RQ plates 30 RQ studies 43 F FAM dye 13 G Gene Expression plots 38 graph settings 29 39 Graph Settings dialog box 39 guidelines assay development 13 21 chemical safety xiii preparing RNA 16 preventing PCR contamination 19 H hazard icons x hazards xv High Capacity CDNA Archive kit 16 52 IMPORTANT description x importing data into RQ plates 24 installing RQ Study Software 49 instrument operational safety xii Instrument tab 26 M master mix PCR 20 materials 4 mode emulation 26 moving and lifting instrument safety x11 MSDSs xiii obtaining viii multiplex PCR 7 N New Detector dialog box 47 normalized reporter 27 O outliers 41 P passive reference 24 27 PCR end point 1 generating data from RQ plates 19 master mix preparing 20 multiplex 7 preventing contamination 19 real time 1 selecting a method 7 singleplex 7 starting an RQ plate run 26 physical hazards xv Plate view 28 plate RQ See RQ plates Plus Minus
74. try Guide Chemistry Step Reagent Part Number TaqMan RT High Capacity cDNA Archive Kit 4322171 t RE JERES PCR TaqMan Universal PCR Master Mix 4304437 SYBR Green I PCR SYBR Green Master Mix 4309155 frue a RT and PCR SYBR Green RT PCR Reagents 4310179 The sample experiment seeks to determine the expression levels of specific genes it requires probes that can bind to specific DNA sequences Premade probes and primers for all the genes of interest were available from the Assays on Demand product line which uses TaqMan chemistry Two step RT PCR was performed using the reagents recommended for TaqMan reagent or kit based chemistry in the table above Notes 12 Relative Quantification Getting Started Guide for 7000 v1 1 Choosing the Probes and Primers About Reagent Configurations for RT PCR Choosing the Probes and Primers You must choose probe and primer sets for both your target and endogenous control sequences Applied Biosystems provides three options for choosing primers and probes Assays on Demand Gene Expression Products Provide you with optimized ready to use TaqMan 5 nuclease assays for human mouse or rat transcripts For information on available primer probe sets go to http www appliedbiosystems com and click the Assays on Demand Gene Expression Products link on the right hand column e Assays by Design Service Designs synthesizes formulat
75. ts where the targets and endogenous controls are amplified in the same well the probe for the target is typically labeled with FAM dye and that for the endogenous control with VIC dye In the sample experiment all target probes were labeled with FAM dye the endogenous control was also labeled with FAM dye GAPDH FAIV The following table lists the gene symbol gene name and Applied Biosystems Assay ID number provided on the Web site for five of the genes studied in the sample experiment plus the endogenous control Gene Symbol Gene Name Assay ID ff ACVR1 acrosomal vesicle protein Hs00153836 m1 ACVR2 activin A receptor type II Hs00155658_m1 CCR2 chemokine C C motif receptor 2 Hs00174150_m1 CD3D CD3D antigen delta polypeptide TiT3 complex Hs00174158_m1 FLT4 fms related tyrosine kinase 4 Hs00176607 m1 GAPDH glyceraldehyde 3 phosphate dehydrogenase Hs99999905 m1 Notes Relative Quantification Getting Started Guide for 7000 v1 1 13 Have you e Selected the PCR method e Designated the four essential components e Selected an SDS chemistry amp reagent configuration e Designed primers and probes Do you have purified aS No Isolate total RNA using page 19 these guidelines Design an RQ experiment according to guidelines in chapter 2 total RNA Yes Is the concentration of RNA within the No recommended range page 16 Adjust RNA concentration
76. ur replicates of each sample and endogenous control are performed to ensure statistical significance following table Note The sample RQ experiment requires a separate plate for each of the three tissues because of the number of genes being studied Experiments can also be designed so that several samples are amplified on the same plate as shown in the In the sample RQ experiment each plate contains a single In experiments where multiple sample types are on the sample type tissue The endogenous control for each same plate an endogenous control for each sample type tissue is on the same plate as the targets for that tissue must also be included on the same plate Liver Kidney Bladder 000000000000 m H N 000000000000 eoeeeeeeccoe 0008 80808 0808 Live 00000000000 Soe See SIOIS18 Samples 0000 8080000080 0000000006086 ore SISIOIS O00 Samples 0900060000 e 0 00606060000 000000000000 00000000000 101010 101010 O Endogenous 0 00080008000080 GAPDH T Controls 000 OO 0 0 OO O O 0 Endogenous Controls GAPDH 000000000080 000000000080 SIGIOIS CIOTOTO G1OIS Kine e 060060600000 Samples 00000000000 000000000080 000000000080 101010 0101010 0 OT Endogenous Controls GAPDH 2000000000080 0000000000080 SISISIS SISISIS CTOTOTO Bladder ororo OOOO OOOO
77. wing table describes the safety symbols that may be displayed on Applied Biosystems instruments Each symbol may appear by itself or in combination with text that explains the relevant hazard see Safety Labels on Instruments on page xii These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Symbol Description Indicates that you should consult the manual for further information and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution Indicates the presence of a laser inside the instrument and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution gt b B gt gt Relative Quantification Getting Started Guide for 7000 v1 1 XI Safety and EMC Compliance Information Safety Labels on Instruments Safety Labels on Instruments The following CAUTION WARNING and DANGER statements may be displayed on Applied Biosystems instruments in combination with the safety symbols described in the preceding section English Francais CAUTION Hazardous chemicals Read the Material Safety Data Sheets MSDSs before handling ATTENTION Produits chimiques dan
78. xperiment were obtained from the Assays on Demand product line and were provided as a 20X Gene Expression Assay Mix The PCR master mix was prepared as follows according to guidelines specified in the product insert that comes with all AOD and ABD products Reaction Component Volume uL Final Concentration Per Sample TaqMan Universal PCR Master Mix 2X 25 0 1X 20X Assays on Demand Gene 2 5 1X Expression Assay Mix contains forward and reverse primers and labeled probe cDNA sample 5 0 50 ng for the 50 uL reaction Nuclease free water 17 5 Total 50 0 The reactions were kept on ice until the plate was loaded on the 7000 SDS instrument Notes Relative Quantification Getting Started Guide for 7000 v1 1 21 Chapter 4 Generating Data from RQ Plates Creating an RQ Plate Document Creating an RQ Plate Document Summary An RQ Plate document is an SDS document that stores data collected from an RQ run for a single plate there must be one RQ Plate document for every RQ plate RQ Plate documents also store other information about the run including sample names and detectors IMPORTANT You cannot change the data collected during the run However the 7000 SDS instrument software allows you to change sample names and detectors even after a run has been completed A plate document appears as a three tabbed pane in the software window Each tab relate

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