Applied Biosystems 7500 Getting Started Manual
  1. Manuals
  2. Brands
  3. Applied Biosystems Manuals
  4. Laboratory Equipment
  5. 7500
  6. Getting started manual

Applied Biosystems 7500 Getting Started Manual

Fast real-time pcr system
Hide thumbs Also See for 7500:
Table of Contents

Advertisement

Quick Links

See also: Installation and Maintenance Manual
Applied Biosystems 7300/7500/7500 Fast
Real-Time PCR System
Relative Quantification
Getting Started Guide
Introduction and
Example
RQ Experiment
Designing an
RQ Experiment
Performing
Primer Extended on mRNA
5'
3'
Reverse
5'
cDNA
Reverse
Primer
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand
3'
5'
cDNA
Transcription
Generating Data
from RQ Plates –
STANDARD
STANDARD
STANDARD
7300 or Standard
7500 System
Generating Data
from RQ Plates –
FAST
FAST
FAST
7500 Fast System
Analyzing Data in
an RQ Study

Advertisement

Table of Contents
loading
Need help?

Need help?

Do you have a question about the 7500 and is the answer not in the manual?

Questions and answers

Related Manuals for Applied Biosystems 7500

Summary of Contents for Applied Biosystems 7500

  • Page 1 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Introduction and Relative Quantification Example RQ Experiment Getting Started Guide Designing an RQ Experiment Performing Primer Extended on mRNA 5′ 3′ Reverse 5′ cDNA Reverse Primer Oligo d(T) or random hexamer Synthesis of 1st cDNA strand 3′...
  • Page 2 NOTICE TO PURCHASER: The Applied Biosystems 7300, 7500 and 7500 Fast Real-Time PCR Systems are real-time thermal cyclers covered by US p atents and corresponding claims in their non-US counterparts, owned by Appl ied Biosystems . No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5’...

  • Page 3: Relative Quantification Experiment Workflow

    Create a new Configure Analyze and If necessary, Performing Chapter 6 baseline and Plate document, an RQ Study RQ Study document analysis settings view results omit samples threshold if desired Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 4 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 5: Table Of Contents

    About the 7300/7500/7500 Fast System ........
  • Page 6 Exporting RQ Study Data ..........78 Appendix A Creating Detectors Appendix B Example RQ Experiment References Index Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 7: Preface

    • A general understanding of hard drives and data storage, file transfers, and copying and pasting. • Networking experience, if you want to integrate the 7300/7500/7500 Fast system into your existing laboratory data flow system. This guide uses the following conventions: Text Conventions •...
  • Page 8 Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems. This service is free and available 24 hours a day.

  • Page 9: How To Obtain More Information

    Preface How to Obtain More Information How to Obtain More Information For more information about using the 7300/7500/7500 Fast system, refer to the Related Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Online Help or the Documentation documents shown below. Online Help...

  • Page 10: How To Obtain 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 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 11: Introduction

    Quantification Transcription Generating Generating Data from Data from STANDARD TANDAR RQ Plates RQ Plates Standard Fast Performing an RQ Study About See page 3 RQ Experiments Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 12: About The 7300/7500/7500 Fast System

    PCR applications (such as relative quantification) in fewer than 40 minutes. The 7300/7500/7500 Fast system allows the user to perform several assay types using Relative plates or tubes in the 96-well format. This guide describes the relative quantification Quantification (RQ) assay type.

  • Page 13: About Rq Experiments

    AQ assay type and consult the Real-Time PCR Systems Chemistry Guide for details on how to set up a run and analyze this type of assay. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 14 The figure below shows a representative amplification plot and includes some of the terms defined in the previous table. Sample Threshold n – No Template Control Baseline Cycle Number Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 15 RQ experiment setup that you can use as a quick-start procedure to familiarize yourself with the RQ workflow. Detailed steps in the RQ workflow are described in the Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 16 Experiment boxes that provide details for some of the related steps in the example experiment. Refer to Appendix B, “Example RQ Experiment,” page 81 for more information. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 17: Designing An Rq Experiment

    TANDAR Select one-step or See page 12 RQ Plates RQ Plates Standard Fast two-step RT-PCR Performing an RQ Study Choose the probes See page 14 and primers Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 18: Selecting The Pcr Method

    The singleplex PCR method is used in the example experiment because: • The number of targets to be amplified (23 genes, plus one endogenous control) is large • Optimization and validation requirements are reduced for singleplex experiments Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 19: Specifying The Components Of An Rq Experiment

    For more information about these requirements, refer to the Real-Time PCR Systems Chemistry Guide. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 20 Liver_96Plate.eps Endogenous Endogenous controls (GAPDH) controls (GAPDH) GR2323 GR2322 GR2324 Kidney Regulus samples Kidney_96Plate.eps Endogenous controls (GAPDH) GR2324 GR2325 Bladder Regulus samples Bladder_96Plate.eps Endogenous controls (GAPDH) GR2325 Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 21: Selecting The Chemistry

    Chapter 2 Designing an RQ Experiment Selecting the Chemistry Selecting the Chemistry Applied Biosystems offers two types of chemistries that you can use to detect PCR About products on real-time instruments, as explained in the following table. Both TaqMan Chemistries ®...

  • Page 22: Selecting One- Or Two-Step Rt-Pcr

    ® However, you cannot use the carryover prevention enzyme, AmpErase UNG, with one-step RT-PCR. For more information about UNG, refer to the Real-Time PCR Systems Chemistry Guide. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 23 Gene Expression Assays product line, which uses TaqMan reagent-based chemistry. Two-step RT-PCR is performed using the reagents recommended for TaqMan reagent- or kit-based chemistry in the table above. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 24: Choosing The Probes And Primers

    For multiplex experiments, the probe for the target is typically labeled with FAM dye and ® that for the endogenous control with VIC dye. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 25 In the example experiment, all target probes are labeled with FAM dye; the endogenous control is also labeled with FAM dye. The following table provides the gene symbol, gene name, and Applied Biosystems Assay ID number (provided on the Web site) for five of the genes studied in the example experiment, plus the endogenous control.
  • Page 26 Chapter 2 Designing an RQ Experiment Choosing the Probes and Primers Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 27: Performing Reverse Transcription

    Transcription Generating Generating Data from Data from STANDARD TANDAR RQ Plates RQ Plates Standard Fast Performing an RQ Study Convert See page 19 total RNA to cDNA Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 28: Guidelines For Preparing Rna

    Convert enough total RNA so that the final concentration of total RNA Starting converted to cDNA is 10 to 100 ng in 5 µL for each 50-µL PCR reaction. Concentration of Total RNA Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 29: Converting Total Rna To Cdna

    Step Type Time Temperature 25 ° C HOLD 10 min 37 ° C HOLD 120 min Thermal cycling conditions for one-step RT-PCR are described on page Note: Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 30 The RNA is then converted to cDNA using the thermal cycling parameters for two-step RT-PCR, as described in “Thermal Profile Parameters for RT” on page The cDNA is stored at − 20 ° C until use. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 31: Generating Data From Rq Plates - 7300 Or Standard 7500 System

    Data from Data from STANDARD RQ Plates RQ Plates Fast Standard See page 32 Start the run Performing an RQ Study See page 33 View RQ plate data Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 32: Before You Begin

    Check that background and pure-dye runs have been performed regularly to ensure optimal performance of the 7300/7500 system. For more information about calibrating the 7300/7500 system, refer to the Online Help located within the software by clicking Preparing the PCR Master Mix The second step (PCR) in the two-step RT-PCR procedure is amplifying the cDNA, ®...

  • Page 33: Preparing The Reaction Plate

    Additionally, every plate must include an endogenous control for every sample type on the plate. Into each well of the reaction plate, add 50 µL of the appropriate PCR master mix. Keep the reaction plates on ice until you are ready to load them into the 7300/7500 system. Notes...
  • Page 34 GR2323 GR2325 GR2324 Kidney Regulus samples Kidney_96Plate.eps Endogenous controls (GAPDH) GR2324 The reactions are kept on ice until the plates are loaded on the 7300/7500/7500 Fast system. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 35: Creating A Relative Quantification (Rq) Plate Document

    Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Creating a Relative Quantification (RQ) Plate Document Creating a Relative Quantification (RQ) Plate Document An RQ Plate document stores data collected from an RQ run for a single plate. There Overview must be one RQ Plate document for every RQ plate.
  • Page 36 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Creating a Relative Quantification (RQ) Plate Document Creating an RQ Plate Document You can enter sample information into a new plate document, import sample information from existing plate documents, or use a template document to set up new plate documents.
  • Page 37 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Creating a Relative Quantification (RQ) Plate Document Select detectors to add to the plate document. Click to select a detector. (Ctrl-click to select multiple detectors.) If no detectors are...
  • Page 38 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Creating a Relative Quantification (RQ) Plate Document Enter the sample names. In the Well Inspector, click a well or click- drag to select replicate wells.
  • Page 39 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Creating a Relative Quantification (RQ) Plate Document Sample Experiment In the example RQ experiment, the samples for each of the three tissues (liver, kidney, and bladder) are loaded on three separate plates.

  • Page 40: Specifying Thermal Cycling Conditions And Starting The Run

    Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Specifying Thermal Cycling Conditions and Starting the Run Specifying Thermal Cycling Conditions and Starting the Run If you selected the two-step RT-PCR method for your RQ experiment (recommended), Default Thermal you have already completed the RT step and are ready to PCR amplify cDNA.
  • Page 41 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Specifying Thermal Cycling Conditions and Starting the Run If you select the one-step RT-PCR method, cDNA generation and amplification take Thermal Cycling place simultaneously at this point in the workflow.
  • Page 42 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Specifying Thermal Cycling Conditions and Starting the Run To specify thermal cycling conditions and start the run: Select the Instrument tab. By default, the standard PCR conditions for the PCR step of the two-step RT-PCR method are displayed.

  • Page 43: Analyzing And Viewing Rq Plate Data

    Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Analyzing and Viewing RQ Plate Data Analyzing and Viewing RQ Plate Data To analyze RQ Plate data after the run, click or select Analysis > Analyze. The SDS Starting the Software 1.3.1 mathematically transforms the raw fluorescence data to establish a...
  • Page 44 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Analyzing and Viewing RQ Plate Data Displays the results data of each well, including the: Plate Tab • Sample name and detector task and color for each well •...
  • Page 45 Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Analyzing and Viewing RQ Plate Data Displays the complete spectral contribution of each dye in a selected well over the Component Tab duration of the PCR run. Only the first selected well is shown at one time.

  • Page 46: Exporting Rq Plate Data

    Chapter 4 Generating Data from RQ Plates – 7300 or Standard 7500 System STANDARD STANDARD STANDARD Exporting RQ Plate Data Exporting RQ Plate Data You can export numeric data from RQ plates into text files, which can then be imported into spreadsheet applications such as Microsoft Excel.

  • Page 47: Generating Data From Rq Plates - 7500 Fast System

    Data from FAST STANDARD TANDAR RQ Plates RQ Plates Standard Fast See page 51 Start the run Performing an RQ Study See page 52 View RQ plate data Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 48: Before You Begin

    7300/7500 system must use standard TaqMan Universal PCR Master Mix (2x) for an approximately 2 hour run time. Users of the 7500 Fast System can choose either the TaqMan Universal PCR Master Mix (2x) or TaqMan Fast Universal PCR Master Mix (2x);...
  • Page 49 50 to 900 nM Reverse primer 50 to 900 nM ® TaqMan probe 50 to 250 nM cDNA sample 10 to 100 ng Nuclease-free water — Total 20.0 — Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 50: Preparing The Reaction Plate

    FAST Preparing the Reaction Plate Preparing the Reaction Plate Make sure that you use the Optical 96-Well Fast Plate on the 7500 Fast Fast vs. IMPORTANT! System. Standard plates will not function properly and may be crushed when using the Standard Plates 96-Well Fast Block.
  • Page 51 Ensure all reaction is positioned correctly in the bottom of the well before IMPORTANT! starting a run. Failure to do so will impact the quality of data. Place the reaction plates on ice until you are ready to load them into the 7500 Fast system. Notes...
  • Page 52 Endogenous controls (GAPDH) GR2324 The reactions are kept on ice until the plates are loaded on the 7500 Fast system. Note: To ensure optimal results, run the reaction plate as soon as possible after completing the reaction setup. If you cannot run a reaction plate within 2 hours after completing the reaction setup, refrigerate or freeze the reaction plate until you can load and run it on the 7500 Fast instrument.

  • Page 53: Creating A Relative Quantification (Rq) Plate Document

    Plate Document plate documents. This section describes setting up new plate documents. Refer to the Online Help for information about importing sample information or using template documents. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 54 The information stored in AQ and RQ Plate documents is not interchangeable. Enter a name in the Default Plate Name field, or accept the default. Click Next >. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 55 Click OK. The SDS Software 1.3.1 creates the plate document and displays the Well Inspector. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 56 Note: information (sample name, detector, task) after a run is complete, if necessary. Close the Well Inspector. Verify the information on each well in the Setup tab. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 57 The figure below shows the example RQ Plate document after sample names, detectors, and detector tasks are assigned for each well in the liver plate. Sample name Detector task and color Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 58: Specifying Thermal Cycling Conditions And Starting The Run

    Specifying Thermal Cycling Conditions and Starting the Run Run assays using Fast thermal cycling conditions. Running Assays Using Fast • Applied Biosystems has verified the performance of Fast thermal cycling and the Thermal Cycling TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, for Conditions quantitative applications only and not for endpoint applications, such as allelic discrimination (SNP Genotyping or Plus/Minus Assays).
  • Page 59 Fast Thermal Cycling Conditions (Fast 7500 users only) Enzyme Activation Melt Anneal/Extend 2) PCR Step 95 ° C 3 sec @ 95 ° C 30 sec @ 60 ° C Fast Conditions 0.20 Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 60 If you wish to enable Expert IMPORTANT! Mode continue to step 3. Otherwise skip to step Click the Expert Mode checkbox. Click the Select/View Filters button. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 61 After the run, a message indicates whether or not the run is successful. All data generated during the run is saved to the RQ Plate document that you saved in step Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 62: Analyzing And Viewing Rq Plate Data

    • To adjust graph settings, double-click the y- or x-axes of a plot to display the Graph Settings dialog. The adjustable settings depend on which plot you are viewing. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 63 Displays the complete spectral contribution of each dye in a selected well over the Component Tab duration of the PCR run. Only the first selected well is shown at one time. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 64 After the software analyzes data, the Analyze button is disabled ( ). Whenever Note: you change a setting that requires reanalysis, the Analyze button is enabled ( Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 65: Exporting Rq Plate Data

    Enter a file name for the export file. The name of the dialog box depends on Note: the type of data you want to export. Click Save. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 66: Troubleshooting

    Evaporation Make sure that the reaction plate is sealed completely, especially around the edges. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 67 3. If you do not obtain acceptable performance by increasing both the annealing/extension temperature and time, assay reoptimization may be required. Refer to the Real-Time PCR Systems Chemistry Guide (PN 4378658) for more information. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 68 Chapter 5 Generating Data from RQ Plates – 7500 Fast System FAST FAST FAST Troubleshooting Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 69: Analyzing Data In An Rq Study

    TANDAR RQ Plates RQ Plates Standard Fast If necessary, See page 75 omit samples Performing Export AQ See page 78 an RQ Study Plate document, if desired Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 70: Creating An Rq Study Document

    • Set baseline and threshold values and RQ (You can perform these operations in RQ Min/Max Confidence Levels. Plate documents.) • Omit individual wells or sample replicates. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 71 The SDS Software 1.3.1 will reject a plate if it detects any differences. (The first plate added to the study serves as the reference plate against which other plates are compared.) Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 72 Plate (default), Amplification Plot, and Gene Expression. You can save the RQ Study Note: document now, or wait until after specifying analysis settings and analyzing the data. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 73: Configuring Analysis Settings

    (You can go back to a saved RQ Plate document and change the sample names, if necessary.) Select the Endogenous Control Detector. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 74 Analysis Settings dialog box, refer to the Online Help. After the analysis, verify that the baseline and threshold were called correctly for each detector, as explained in the following section. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 75: Adjusting The Baseline And Threshold

    68 for each of the detectors. and Threshold Determination The following amplification plots show the effects of baseline and threshold settings. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 76 The amplification curve begins too far to the right of the maximum baseline. Increase the End Cycle value. Baseline Set Too High The amplification curve begins before the maximum baseline. Decrease the End Cycle value. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 77 The standard error is significantly higher than that for a plot where the threshold is set correctly. Drag the threshold bar down into the exponential phase of the curve. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 78 When manually adjusting baseline and threshold settings, you can select Note: only one detector at a time. If you select multiple detectors, the Analysis Settings section and the threshold bar are disabled. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 79 The bar turns red, indicating that the threshold has been changed. Click or select Analysis > Analyze to reanalyze the data using the adjusted baseline and threshold values. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 80: Analyzing And Viewing The Results Of The Rq Study

    • 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 for GAPDH. − ∆∆CT • Fold-expression changes are calculated using the equation 2 Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 81 Study” on page 75 for more information). If there is a data point at Ct0, this point is Note: undetermined, the data point is not actually Ct = 0. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 82 Gene Expression Plot Orientation: Detector Detectors are plotted on the x-axis, and each bar shows the detector value of a single sample. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 83 The SDS Software 1.3.1 calculates the error bars based on the RQMin/Max Confidence Level in the Analysis Settings dialog box (see page 63). Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 84: Reanalyzing An Rq Study

    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. Calibrator Gene Expression Plot Liver Kidney Bladder Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 85: Omitting Samples From A Study

    All samples that use this detector are displayed in the RQ Samples grid. In the RQ Samples grid, click to select the samples to display in the Amplification Plot. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 86 Verify the uniformity of each replicate population by comparing the groupings of C values for the wells that make up the set. Good clustering of replicate data. No outliers. Potential outlier. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 87 Repeat steps 3 to 8 for other detectors you want to screen. Select Omit. This is an enlarged version showing that the outlier is The outlier is removed removed during during analysis. analysis. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 88: Exporting Rq Study Data

    Enter a file name for the export file. The name of the dialog box depends on Note: the type of data you want to export. Click Save. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 89: Appendix A Creating Detectors

    (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. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 90 FAM was selected for the reporter dye. Additionally, TaqMan Custom Gene Expression Assays use TaqMan MGB probes, which use nonfluorescent quenchers. “None” is selected for the quencher dye. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 91 Data are generated by running three RQ plates, one for each tissue. All three plates are analyzed in an RQ study, with the liver samples serving as the calibrator. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 92: Appendix B Example Rq Experiment

    RT reactions per tissue. Extra volume (enough for one additional RT reaction per tissue) is included to account for pipetting losses, as well as extra cDNA for archiving. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 93 § 24 master mixes are prepared, one for each of 23 genes plus the endogenous control. Volume for five reactions (4 replicates plus gloves. extra) to account for pipetting losses. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 94 Regulus samples ready to load them into the Kidney_96Plate.eps 7300/7500/7500 Fast system. Endogenous controls (GAPDH) GR2324 GR2325 Bladder Regulus samples Bladder_96Plate.eps Endogenous controls (GAPDH) GR2325 Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 95 Finish. You cannot add RQ plates to RQ studies unless you have specified sample names, as indicated in the message shown to the right. Click OK. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 96 For more information about omitting wells, refer to the Online Help. The figure on the right shows a completed plate set up. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 97 Click Add Plates to add plates to the study, then click Open. You can add up to 10 RQ plates to an Note: RQ study. Click Finish. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 98 As shown in the figure on the right where liver is used as the calibrator, expression levels of CCR2 are greater in the liver than in the kidney or bladder tissues of this individual. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 99 Method. Methods 25:402–408. Saiki, R.K., Scharf, S., Faloona, F., et al. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354. Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 100: References

    References Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 101 RQ studies definition CAUTION, description selecting for RQ studies deviation, standard cDNA generating display options 33, 52, 71, storing documentation, related See also reverse transcription Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...

  • Page 102: Index

    RQ. See RQ plates Gene Expression plots plateau phase of amplification curve graph settings 33, 52, 71, plot appearance 33, 52, 71, guidelines Primer Express Software preparing RNA primers Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 103 26, reanalyzing data 35, text conventions results 33, RQ Plate documents 25, thermal cycling conditions Spectra view 34, default for PCR 30, starting a run 32, Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 104 WARNING, description Well Information tab wells, replicate width, bars width, lines 33, 52, 71, workflow, RQ experiment overview x-axis 33, 52, 71, y-axis 33, 52, 71, Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide...
  • Page 106 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.

This manual is also suitable for:

7300

Table of Contents