High-throughput real-time quantitative reverse transcription PCR

Curr Protoc Mol Biol. 2006 Feb;Chapter 15:Unit 15.8. doi: 10.1002/0471142727.mb1508s73.

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 DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomedical Research / methods*
  • DNA Primers
  • Indicators and Reagents
  • Nucleic Acid Probes / chemical synthesis
  • RNA / standards*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards

Substances

  • DNA Primers
  • Indicators and Reagents
  • Nucleic Acid Probes
  • RNA