Ingeniero Bioquimico

Applications Guide

Real-Time PCR Applications Guide

table of contents

Table of Contents
1. Overview of Real-Time PCR 1.1 Key Concepts of Real-Time PCR 1.1.1 What Is Real-Time PCR? 1.1.2 How Real-Time PCR Works 1.1.3 Hallmarks of an Optimized qPCR Assay 2. Getting Started 2.1 General Considerations 2.2 Experimental Design Considerations for qPCR 2.2.1 Singleplex or Multiplex? 2.2.2Chemistry Selection 2.2.2.1 DNA-Binding Dyes (SYBR Green I) 2.2.2.2 Fluorescent Primer- and Probe-Based Chemistries 2.3 Design and Optimization of SYBR Green I Reactions 2.3.1 Primer and Amplicon Design 2.3.2 Assay Validation and Optimization 2.3.2.1 Annealing Temperature Optimization 2.3.2.2 Assay Performance Evaluation Using Standard Curves 2.4 Design and Optimization of TaqMan Probe Reactions 2.4.1Primer and Probe Design 2.4.2 Assay Validation and Optimization 3. Multiplexing Considerations 3.1 Primer and Probe Design for Multiplexing 3.2 Selection of Reporters and Quenchers for Multiplexing 3.3 Optimization of Individual Assays Before Multiplexing 3.4 Validation of Multiplex Assays 3.5 Optimization of Multiplex Assays 2 2 2 3 4 8 8 9 9 9 10 12 19 19 20 20 22 23 23 24 28 28 29 29 29 30table of contents i

table of contents

4. Real-Time qPCR Data Analysis 4.1 Absolute Quantification 4.1.1 When Should Absolute Quantification Be Used? 4.1.2 Absolute Quantification Using a Standard Curve 4.2 Relative Quantification 4.2.1 When Should Relative Quantification Be Used? 4.2.2 Relative Quantification Normalized Against Unit Mass 4.2.3 Relative Quantification Normalized to aReference Gene 4.2.3.1 The 2–∆∆CT (Livak) Method 4.2.3.2 The ∆CT Method Using a Reference Gene 4.2.3.3 The Pfaffl Method 5. Gene Expression Analysis 5.1 Experimental Design 5.2 RNA Isolation 5.2.1 Sample Collection 5.2.2 RNA Extraction 5.2.3 Analyzing Nucleic Acid Quantity and Quality 5.3 cDNA Template Preparation (Reverse Transcription) 5.4 qPCR Assay Development 5.5 Experimentation 5.5.1 ReactionComponents for Multiplex Assay 5.5.2 Cycling Protocol 5.6 Gene Expression Data Analysis 6. Genotyping/Allelic Discrimination 6.1 Experimental Design 6.2 Primer and Probe Design Using TaqMan Probes 6.3 DNA Extraction and Sample Preparation 6.4 Reaction Components When Using TaqMan Probes 6.5 Optimization 6.6 Cycling Protocol Using TaqMan Probes 6.7 Allelic Discrimination Data Analysis 6.7.1 Validationof Allelic Discrimination Assay 6.7.2 Genotype Assignments

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table of contents ii

table of contents

7. Genetically Modified Organism (GMO) Detection 7.1 Experimental Design 7.2 DNA Extraction and Sample Preparation for GMO Detection 7.3 GM Soy Detection Using a Singleplex SYBR Green I qPCRAssay 7.3.1 Reaction Components 7.3.2 Cycling Protocol 7.3.3 Data Analysis 7.4 GM Soy Detection Using a Multiplex TaqMan qPCR Assay 7.4.1 Reaction Components 7.4.2 Cycling Protocol 7.4.3 Data Analysis 8. Product Guide

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table of contents iii

Overview of Real-Time PCR
Key Concepts of Real-Time PCR What Is Real-Time PCR? How Real-Time PCR Works Hallmarksof an Optimized qPCR Assay 2 2 3 4

overview of real-time PCR
key concepts of real-time PCR

1. Overview of Real-Time PCR
Nucleic acid amplification and detection are among the most valuable techniques used in biological research today. Scientists in all areas of research — basic science, biotechnology, medicine, forensic science, diagnostics, and more — rely on these methods for a widerange of applications. For some applications, qualitative nucleic acid detection is sufficient. Other applications, however, demand a quantitative analysis. Choosing the best method for your application requires a broad knowledge of available technology. This guide provides an introduction to many of the technical aspects of real-time PCR. It includes guidelines for designing the best real-time PCR...