Quantitative RT-PCR: Pitfalls and potential

Willard M. Freeman, Stephen J. Walker, Kent E. Vrana

Research output: Contribution to journalReview articlepeer-review

890 Scopus citations


Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it was tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real- time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.

Original languageEnglish (US)
Pages (from-to)112-125
Number of pages14
Issue number1
StatePublished - 1999

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • General Biochemistry, Genetics and Molecular Biology


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