Standardized RT-PCR and the standardized expression measurement center

Methods Mol Biol. 2004;258:13-41. doi: 10.1385/1-59259-751-3:13.

Abstract

Standardized reverse transcriptase polymerase chain reaction (StaRT-PCR) is a modification of the competitive template (CT) RT method described by Gilliland et al. StaRT-PCR allows rapid, reproducible, standardized, quantitative measurement of data for many genes simultaneously. An internal standard CT is prepared for each gene, cloned to generate enough for 10(9) assays and CTs for up to 1,000 genes are mixed together. Each target gene is normalized to a reference gene to control for cDNA loaded in a standardized mixture of internal standards (SMIS) into the reaction. Each target gene and reference gene is measured relative to its respective internal standard within the SMIS. Because each target gene and reference gene is simultaneously measured relative to a known number of internal standard molecules in the SMIS, it is possible to report each gene expression measurement as a numerical value in units of target gene cDNA molecules/ 10(6) reference gene cDNA molecules. Calculation of data in this format allows for entry into a common databank, direct interexperimental comparison, and combination of values into interactive gene expression indices.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA, Complementary / analysis
  • Gene Expression*
  • Humans
  • RNA, Messenger / analysis
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / standards*

Substances

  • DNA, Complementary
  • RNA, Messenger