Unit

UNIT 15.8 High-Throughput Real-Time Quantitative Reverse Transcription PCR

  1. Angie L. Bookout,
  2. Carolyn L. Cummins,
  3. David J. Mangelsdorf

Published Online: 1 AUG 2005

DOI: 10.1002/0471142727.mb1508s71

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Bookout, A. L., Cummins, C. L. and Mangelsdorf, D. J. 2005. High-Throughput Real-Time Quantitative Reverse Transcription PCR. Current Protocols in Molecular Biology. 15:15.8.

Author Information

  1. Howard Hughes Medical Institute University of Texas Southwestern Medical Center, Dallas, Texas

Publication History

  1. Published Online: 1 AUG 2005
  2. Published Print: JUL 2005

This is not the most recent version of the article. View current version (1 FEB 2006)

Abstract

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected ΔCt method, and the comparative cycle time, or ΔCt method.

Keywords:

  • QPCR;
  • quantitative PCR;
  • real-time PCR;
  • RNA expression analysis;
  • TaqMan;
  • SYBR Green;
  • ABI 7900HT;
  • reverse transcriptase PCR