Unit

UNIT 10.3 Real-Time PCR

  1. Dean Fraga1,
  2. Tea Meulia2,
  3. Steven Fenster3

Published Online: 11 FEB 2014

DOI: 10.1002/9780470089941.et1003s08

Current Protocols Essential Laboratory Techniques

Current Protocols Essential Laboratory Techniques

How to Cite

Fraga, D., Meulia, T. and Fenster, S. 2014. Real-Time PCR. Current Protocols Essential Laboratory Techniques. 10:10.3:10.3.1–10.3.40.

Author Information

  1. 1

    College of Wooster, Wooster, Ohio

  2. 2

    Ohio Agricultural Research and Development Center, Wooster, Ohio

  3. 3

    Fort Lewis College, Durango, Colorado

Publication History

  1. Published Online: 11 FEB 2014

Abstract

Real-time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished using specific as well as nonspecific fluorescent probes. Real-time PCR is often used in the quantification of gene expression levels. Prior to using real-time PCR to quantify a target message, care must be taken to optimize the RNA isolation, primer design, and PCR reaction conditions so that accurate and reliable measurements can be made. This short overview of real-time PCR discusses basic principles behind real-time PCR, some optimization and experimental design considerations, and how to quantify the data generated using both relative and absolute quantification approaches. Useful Web sites and texts that expand upon topics discussed are also listed. Curr. Protoc. Essential Lab. Tech. 8:10.3.1-10.3.40. © 2014 by John Wiley & Sons, Inc.

Keywords:

  • quantifying gene expression;
  • real-time PCR (polymerase chain reaction);
  • cDNA;
  • PCR primer design;
  • Taq polymerase;
  • SYBR Green;
  • PCR