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Introduction Designing an RQ Experiment Performing Reverse Transcription Genera

Introduction Designing an RQ Experiment Performing Reverse Transcription Generating Data from RQ Plates – 7300/7500 System Generating Data from RQ Plates – 7500 Fast System Analyzing Data in an RQ Study Primer Extended on mRNA 5′ 3′ Reverse Primer 5′ Synthesis of 1st cDNA strand cDNA Oligo d(T) or random hexamer 3′ 5′ cDNA STANDARD STANDARD STANDARD FAST FAST FAST Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantitation Using Comparative CT Getting Started Guide © Copyright 2006, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF . NOTICE TO PURCHASER: The Applied Biosystems 7300, 7500 and 7500 Fast Real-Time PCR Systems are real-time thermal cyclers covered by US patents and corresponding claims in their non-US counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5′ nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TRADEMARKS: AB (Design), Applied Biosystems, ABI PRISM, Primer Express, and VIC are registered trademarks and Applera, FAM, MicroAmp, MultiScribe, ROX, TAMRA, and Tempus are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or other countries. AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. SYBR Green is a registered trademark of Molecular Probes, Inc. Excel, Microsoft, PowerPoint, and Windows are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Part Number 4347824 Rev. F 06/2010 Chapter 2 Select the PCR Method Specify the Components of an RQ Experiment Select the Chemistry Select One- or Two-Step RT-PCR Choose the Probes and Primers Chapter 3 Guidelines for Preparing RNA Convert Total RNA to cDNA Primer Extended on mRNA 5´ 3´ Reverse Primer 5´ Synthesis of 1st cDNA strand cDNA Oligo d(T) or random hexamer 3´ 5´ cDNA Create an RQ Study Document Configure Analysis Settings Adjust the Baseline and Threshold Analyze and View the Results of the RQ Study Reanalyze an RQ Study Omit Samples from a Study Export RQ Study Data Chapter 6 Chapter 5 Chapter 1 About the 7300/7500/7500 Fast System About Relative Quantitation About RQ Experiments Before You Begin Prepare the PCR Master Mix Create a Relative Quantitation (RQ) Plate Document Prepare the Reaction Plate Specify Thermal Cycling Conditions and Start the Run Analyze and View RQ Plate Data Export RQ Plate Data Before You Begin Prepare the PCR Master Mix Create a Relative Quantitation (RQ) Plate Document Prepare the Reaction Plate Specify Thermal Cycling Conditions and Start the Run Analyze and View RQ Plate Data Exporting RQ Plate Data Troubleshooting Chapter 4 (OR) Designing an RQ Experiment Performing Reverse Transcription Primer Extended on mRNA 5´ 3´ Reverse Primer 5´ Synthesis of 1st cDNA strand cDNA Oligo d(T) or random hexamer 3´ 5´ cDNA Analyzing Data in an RQ Study Introduction Generating Data from RQ Plates Standard Generating Data from RQ Plates Fast STANDARD STANDARD FAST FAST STANDARD STANDARD FAST FAST Relative Quantitation Using Comparative CT Assay Getting Started Guide for 7300/7500/7500 Fast Systems iii Relative Quantitation Experiment Workflow iv Relative Quantitation Using Comparative CT Assay Getting Started Guide for 7300/7500/7500 Fast Systems Contents Relative Quantitation Using Comparative CT Assay Getting Started Guide for 7300/7500/7500 Fast Systems v Relative Quantitation Experiment Workflow iii Preface vii How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Safety xi Safety Alert Words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii General Chemical Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii General Biohazard Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii General Chemical Waste Hazard Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv Obtaining MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv Chapter 1 Introduction 1 About the 7300/7500/7500 Fast System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 About Relative Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 About RQ Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Chapter 2 Designing an RQ Experiment 7 Selecting the PCR Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Specifying the Components of an RQ Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Selecting the Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Selecting One- or Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Choosing the Probes and Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Chapter 3 Performing Reverse Transcription 17 Guidelines for Preparing RNA . . . . . . . . uploads/Geographie/ abi-7300-rq-guide.pdf