Quantitative RT-PCR: Pitfalls and potential

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative appl ications, a comprehensive knowledge of its technical aspects is required. S uccessful quantitative RT-PCR involves correction for experimental variatio ns in individual RT and PCR efficiencies. This review addresses the mathema tics of RT-PCR, choice of RNA standards (internal vs. external) and quantif ication strategies (competitive, noncompetitive and kinetic [real-time] amp lification). Finally, the discussion turns to practical considerations in e xperimental design. It is hoped that this review will be appropriate for th ose undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequency a confusing an contradictory field.