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Related Concept Videos

PCR01:32

PCR

Overview
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
PCR - Polymerase Chain Reaction01:32

PCR - Polymerase Chain Reaction

Overview

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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

Published on: May 22, 2012

Explanatory chapter: quantitative PCR.

Jessica S Dymond1

  • 1The High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Methods in Enzymology
|September 10, 2013
PubMed
Summary
This summary is machine-generated.

Quantitative PCR (qPCR) measures DNA concentration in real-time. This powerful technique is essential for applications like viral load detection and gene expression analysis, serving as a gold standard in molecular biology research.

Keywords:
Amplification efficiencyPrimer designQuantitative PCRSYBR Green I (SG)TaqMan Probes

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Quantitative PCR (qPCR) is a widely used technique for DNA amplification and quantification.
  • It enables precise measurement of target DNA concentrations within a reaction.
  • qPCR is also known as real-time PCR or quantitative real-time PCR.

Purpose of the Study:

  • To provide a comprehensive overview of qPCR techniques.
  • To outline the technical background, available chemistries, and data analysis methods.
  • To offer a framework for experimental design and evaluation in qPCR applications.

Main Methods:

  • Real-time PCR (qPCR) coupled with DNA amplification.
  • Reverse-transcription PCR (RT-PCR) for mRNA expression analysis.
  • Analysis of DNA copy number, viral load, SNP detection, and allelic discrimination.

Main Results:

  • qPCR allows for the accurate calculation of starting template concentrations.
  • It is a critical tool for validating microarray gene expression data.
  • Various technical variations and applications of qPCR have been developed.

Conclusions:

  • qPCR is a versatile and powerful analytical tool in molecular biology.
  • Understanding qPCR methodologies is crucial for effective experimental design.
  • The technique is fundamental for numerous applications in research and diagnostics.