quantitative PCR review
Jan 14, 1999
As you know, quantitative PCR is the basis for viral load testing. I thought you might be interested in a reveiw article from a non-hiv scientist, in which it becomes clear that this methodology has resulted in a body of literature that is both "confusing and contradictory". One of the biggest issues that is ignored by so many scientists who attempt to use this appealing methodology is the requirement for equal amplification efficiency between the competitor and the target templates. It is known that this relationship is sequence dependent, and deviations from equal amplification can result in tremendous differences in the estimated amount of target (due to the exponential process of PCR). How is it that when this method is applied to hiv, which is one of the most variable templates one could imagine, it is always assumed to be accurate?
I hope you find the article interesting, and perhaps will consider the ramifications of using such an assay to make profound medication decisions, often with highly toxic drugs.
Todd Miller, PhD Biochemistry and Molecular Biology *****************************************
Article Title Quantitative RT-PCR: Pitfalls and Potential
Article Abstract Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correciton 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.
Article Authors Freeman, W. M.; Walker, S. J.; Vrana, K. E.
Citation (Review, BioTechniques 26:112-125, January 1999)
Topic Keywords Quantitative RT-PCR; reverse transcription; RNA; mathematics; quantification
Response from Dr. Holodniy
No arguement here regarding the practical limitations of PCR quantitation. Would you have the same critique about the signal amplification of branched DNA technology? MH
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