RT-PCR Protocols

Volume 193 of the series Methods in Molecular Biology pp 83-92

Quantitative RT-PCR

A Review of Current Methodologies
  • Caroline JoyceAffiliated withDepartment of Biochemistry, University Hospital

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The powerful amplification potential of PCR has assured its use in the detection of low-abundance mRNA in cells and tissues. RT-PCR is currently the most sensitive technique available for mRNA detection and quantification. It can accurately quantify genes present at only a few hundred copies per sample, and has become the method of choice for the examination of gene expression. The technique consists of two parts: synthesis of cDNA from RNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction (PCR). The method requires very little RNA and differs from Northern blotting because it is somewhat tolerant of degraded RNA, as long as the RNA is intact within the region of interest.