Title RT-PCR Guide.pdf ✅

table of contents table of contents i Table of Contents 1. Overview of Real-Time PCR 2 1.1 Key Concepts of Real-Time PCR 2 1.1.1 What Is Real-Time PCR? 2 1.1.2 How Real-Time PCR Works 3 1.1.3 Hallmarks of an Optimized qPCR Assay 4 2. Getting Started 8 2.1 General Considerations 8 2.2 Experimental Design Considerations for qPCR 9 2.2.1 Singleplex or Multiplex? 9 2.2.2 Chemistry Selection 9 2.2.2.1 DNA-Binding Dyes (SYBR Green I) 10 2.2.2.2 Fluorescent Primer- and Probe-Based Chemistries 12 2.3 Design and Optimization of SYBR Green I Reactions 19 2.3.1 Primer and Amplicon Design 19 2.3.2 Assay Validation and Optimization 20 2.3.2.1 Annealing Temperature Optimization 20 2.3.2.2 Assay Performance Evaluation Using Standard Curves 22 2.4 Design and Optimization of TaqMan Probe Reactions 23 2.4.1 Primer and Probe Design 23 2.4.2 Assay Validation and Optimization 24 3. Multiplexing Considerations 28 3.1 Primer and Probe Design for Multiplexing 28 3.2 Selection of Reporters and Quenchers for Multiplexing 29 3.3 Optimization of Individual Assays Before Multiplexing 29 3.4 Validation of Multiplex Assays 29 3.5 Optimization of Multiplex Assays 30
table of contents table of contents ii 4. Real-Time qPCR Data Analysis 34 4.1 Absolute Quantification 35 4.1.1 When Should Absolute Quantification Be Used? 35 4.1.2 Absolute Quantification Using a Standard Curve 35 4.2 Relative Quantification 37 4.2.1 When Should Relative Quantification Be Used? 37 4.2.2 Relative Quantification Normalized Against Unit Mass 38 4.2.3 Relative Quantification Normalized to a Reference Gene 40 4.2.3.1 The 2–∆∆CT (Livak) Method 41 4.2.3.2 The ∆CT Method Using a Reference Gene 42 4.2.3.3 The Pfaffl Method 43 5. Gene Expression Analysis 46 5.1 Experimental Design 46 5.2 RNA Isolation 47 5.2.1 Sample Collection 47 5.2.2 RNA Extraction 48 5.2.3 Analyzing Nucleic Acid Quantity and Quality 48 5.3 cDNA Template Preparation (Reverse Transcription) 49 5.4 qPCR Assay Development 50 5.5 Experimentation 51 5.5.1 Reaction Components for Multiplex Assay 51 5.5.2 Cycling Protocol 51 5.6 Gene Expression Data Analysis 52 6. Genotyping/Allelic Discrimination 54 6.1 Experimental Design 55 6.2 Primer and Probe Design Using TaqMan Probes 56 6.3 DNA Extraction and Sample Preparation 57 6.4 Reaction Components When Using TaqMan Probes 58 6.5 Optimization 59 6.6 Cycling Protocol Using TaqMan Probes 59 6.7 Allelic Discrimination Data Analysis 59 6.7.1 Validation of Allelic Discrimination Assay 59 6.7.2 Genotype Assignments 72
7. Genetically Modified Organism (GMO) Detection 76 7.1 Experimental Design 77 7.2 DNA Extraction and Sample Preparation for GMO Detection 77 7.3 GM Soy Detection Using a Singleplex SYBR Green I qPCR Assay 78 7.3.1 Reaction Components 78 7.3.2 Cycling Protocol 79 7.3.3 Data Analysis 79 7.4 GM Soy Detection Using a Multiplex TaqMan qPCR Assay 81 7.4.1 Reaction Components 82 7.4.2 Cycling Protocol 82 7.4.3 Data Analysis 82 8. Product Guide 85 table of contents table of contents iii