Quantitative RT-PCR: Pitfalls and potential

被引：837
作者：
Freeman, WM [1 ]
Walker, SJ [1 ]
Vrana, KE [1 ]
机构：
[1] Wake Forest Univ, Bowman Gray Sch Med, Dept Physiol & Pharmacol, Winston Salem, NC 27157 USA
来源：
BIOTECHNIQUES | 1999年 / 26卷 / 01期
关键词：
D O I：
10.2144/99261rv01
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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 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 frequency a confusing an contradictory field.
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页码：112 / +
页数：12
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