Real-time PCR

M Dorak, M Tevfik Dorak, M Dorak, M Tevfik Dorak

• Publisher: Taylor & Francis
• Year: 2007
• eBook ISBN: 9781134183999
• Topic: Medicine
• Subtopic: Biotechnology in Medicine

With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It addresses the most practical aspects of the techniques with the emphasis on 'how to do it in the laboratory'. Keeping with the spirit of the Advanced Methods Series, most chapters provide an experimental protocol as an example of a specific assay.

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Contributors
• Abbreviations
• Preface
• Glossary
• Links to Software and Web Resources Cited
• 1 An Introduction to Real-time PCR
• Protocol 1.1: Treatment of Total RNA with RNase-free DNase I
• Protocol 1.2: Hybrid Protocol for the Isolation of Total RNA
• Protocol 1.3: Two-step Master Mix
• Protocol 1.4: Two-step PCR Master Mix for Probe-based Assays
• Protocol 1.5: Two-step PCR Master Mix – Primer or SYBR® Green I Based Assay
• 2 Data Analysis and Reporting
• 3 Relative Quantification
• 4 Normalization
• 5 High-throughput Primer and Probe Design
• 6 Quantitative Analysis of Ocular Gene Expression
• 7 Quantitative Gene Expression by Real-time PCR: A Complete Protocol
• 8 Real-time PCR Using SYBR® Green
• 9 High-resolution Melting Analysis for Scanning and Genotyping
• 10 Quantitative Analyses of DNA methylation
• Protocol 10.1
• 11 Mitochondrial DNA Analysis
• 12 Real-time Immuno-PCR
• Protocol 12.1: Basic Real-time Immuno-PCR Protocol
• 13 Clinical Microbiology
• Protocol 13.1: Detection of Prevotella Intermedia by Real-time PCR Assay
• 14 Clinical Virology
• Protocol 14.1: Detection of Influenza A by a 5’ Nuclease Real-time RT-PCR Assay
• Protocol 14.2: Detection and Differentiation of Herpes Simplex Virus Types 1 and 2 Using a Hybridization Probe Real-time PCR assay
• Protocol 14.3: Quantitative Analysis of BKV Load by a 5’ Nuclease Real-time PCR (BKV-Tag-qPCR) Assay
• Protocol 14.4: Real-time PCR Internal Control (Niesters, 2004)
• Troubleshooting Guide 14.1: False-negative Result in the WhSl-para3–5N Assay
• Troubleshooting Guide 14.2: Variation in Fluorescent Signal from Positive Specimens in the RSV MGB Assay
• Troubleshooting Guide 14.3: Uncharacteristic Herpes Simplex Virus Type 2 Melting Curves
• Troubleshooting Guide 14.4: Underestimation of BK Viral Load due to Sequence Variation
• 15 Solid Organ Transplant Monitoring
• Protocol 15.1: Monitoring Solid Organ Transplant Recipients for Rejection
• 16 Real-time PCR Applications in Hematology
• Protocol 16.1: Post-transplant MRD Monitoring
• Protocol 16.2: Use of TSPY Repeats on Y-chromosome in Chimerism Studies
• 17 Real-time PCR for Prenatal Diagnosis of Monogenic Diseases Caused by Single Nucleotide Changes – The Example of the Hemoglobinopathies
• Protocol 17.1: Cleaning Chorionic Villi (CVS) by Microscopic Dissection
• Protocol 17.2: Centrifugation of Amniotic Fluid Cell Samples to Collect Amniocytes
• Protocol 17.3: DNA Extraction from Chorionic Villi Samples or Amniocytes Using Commercially Available DNA Purification Columns
• Protocol 17.4: Protocol for Rapidly Screening Multiple β-globin Gene Mutations by Real-time PCR: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes
• Protocol 17.5: Real-time PCR Protocols for Singlecell Genotyping and PGD
• Protocol 17.6: Nested PCR and genotyping of β-globin gene using Real-Time PCR in the LightCycler®
• Protocol 17.7: Monitoring of Contamination by Sizing GABRB3 and D13S314 Polymorphic Microsatellites
• Protocol 17.8: Technical Tips and Troubleshooting when Performing Real-time PCR and Genotyping Using the LightCycler® System 1.0 and 1.5