Quantitative RT-PCR: Pitfalls and potential

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

Research output: Contribution to journalReview article

720 Citations (Scopus)

Abstract

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
JournalBioTechniques
Volume26
Issue number1
StatePublished - Jan 20 1999

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Transcription
Reverse Transcription
Polymerase Chain Reaction
RNA
Design of experiments
Amplification
Mathematics
Kinetics
Research Design
Experiments

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Freeman, W. M., Walker, S. J., & Vrana, K. (1999). Quantitative RT-PCR: Pitfalls and potential. BioTechniques, 26(1), 112-125.
Freeman, Willard M. ; Walker, Stephen J. ; Vrana, Kent. / Quantitative RT-PCR : Pitfalls and potential. In: BioTechniques. 1999 ; Vol. 26, No. 1. pp. 112-125.
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Freeman, WM, Walker, SJ & Vrana, K 1999, 'Quantitative RT-PCR: Pitfalls and potential', BioTechniques, vol. 26, no. 1, pp. 112-125.

Quantitative RT-PCR : Pitfalls and potential. / Freeman, Willard M.; Walker, Stephen J.; Vrana, Kent.

In: BioTechniques, Vol. 26, No. 1, 20.01.1999, p. 112-125.

Research output: Contribution to journalReview article

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Freeman WM, Walker SJ, Vrana K. Quantitative RT-PCR: Pitfalls and potential. BioTechniques. 1999 Jan 20;26(1):112-125.