Lubing Zhou

Use of quantitative polymerase chain reaction to study cellular retinoic acid-binding protein-II mRNA expression in human skin.

Publisher Summary The use of linked reverse transcription–polymerase chain reactions (RT–PCR) to quantitate specific mRNAs is rapidly gaining favor over Northern blot or RNase protection analyses due to its convenience, speed, and sensitivity. However, accurate quantitative results require careful design of proper controls. The system described in this chapter uses a synthetic RNA generated by in vitro transcription from the cDNA of human CRABP-II, with a small deletion in the coding region. Using identical primers, the test sample and internal control amplify competitively in each reaction tube. This strategy avoids problems due to tube-to-tube variations, unequal amplification efficiencies, and the plateau effect that arise with external controls or unrelated internal controls. A titration system is used to quantitate CRABP-II mRNA accurately. A serial dilution of the sample RNA, together with a fixed amount of internal control RNA, is co-reverse transcribed by a CRABP-II-specific primer. An aliquot of each RT reaction is subsequently amplified by PCR. Because the target and control cDNAs amplify with identical efficiencies, the ratio of the two products is cycle number independent, and the products can be allowed to accumulate to high levels. Following polyacrylamide gel electrophoresis (PAGE), the two products are visualized by ethidium bromide staining.