Real-time PCR for mRNA quantitation

Biotechniques. 2005 Jul;39(1):75-85. doi: 10.2144/05391RV01.

Abstract

Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.

Publication types

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

MeSH terms

  • Algorithms*
  • Chemistry Techniques, Analytical / methods*
  • Gene Expression Profiling / methods*
  • RNA, Messenger / analysis*
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Spectrometry, Fluorescence / methods*

Substances

  • RNA, Messenger