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Real-Time Quantitative PCR: Theory and Practice

Nucleic Acids

  1. Gregory L. Shipley

Published Online: 15 SEP 2006

DOI: 10.1002/3527600906.mcb.200500012

Reviews in Cell Biology and Molecular Medicine

Reviews in Cell Biology and Molecular Medicine

How to Cite

Shipley, G. L. 2006. Real-Time Quantitative PCR: Theory and Practice. Reviews in Cell Biology and Molecular Medicine. .

Author Information

  1. The University of Texas Health Science Center, Houston, TX

Publication History

  1. Published Online: 15 SEP 2006

Abstract

Transcript quantification methodology has evolved from Northern blots to RNase protection to competitive RT-PCR. Each of these methods made strides toward greater sensitivity and accuracy in quantification. Since the introduction of the 7700 Sequence Detection System and Taqman® chemistry by Applied Biosystems in 1996, quantitative real-time PCR has become widely accepted as the most sensitive and accurate method for the quantification of both RNA and DNA from a wide variety of sources. Today, there are a number of real-time instruments from a variety of vendors and several detection methodologies and kit chemistries available. At the same time, prices for real-time platforms are falling dramatically. This trend means that the technology is moving from one found primarily in core laboratories to the bench tops of individual investigators.

There are several ways to interrogate a cell for changes induced by artificial or natural agents during a biological process. One way is to look for changes in cellular transcript levels that may indicate downstream changes in the corresponding protein. In another instance, the focus may be on the presence or absence of a viral or bacterial pathogen. In this case, detecting not only the presence but also the degree of infection provides valuable information. Alternatively, looking for the presence or the level of expression from a transgene or the inhibition of expression of an endogenous gene by an RNAi agent may be the question of interest. In all cases, quantitative real-time PCR technology can be utilized to provide the required results. However, successful implementation of the technology requires the user to have a basic background in the theoretical principles of real-time PCR as well as their practical application. The goal of this review is to provide this information.

It will not be possible to cover all aspects of real-time PCR in this review. For a more comprehensive overview of real-time PCR, “A-Z of Quantitative PCR,” edited by Stephen Bustin, is highly recommended.

Keywords:

  • Amplicon;
  • Baseline;
  • Fluorescence Resonance Energy Transfer;
  • Polymerase Chain Reaction;
  • Real-time PCR;
  • Reverse Transcription;
  • Threshold