Quantitation of mRNA by the kinetic polymerase chain reaction assay: a tool for monitoring P-glycoprotein gene expression

Abstract

The overexpression of P-glycoprotein (mdr1) induces the phenotype of multidrug resistance to many chemically unrelated drugs. Absolute mdr1 mRNA levels in tissues, neoplasms and cell lines from various rodents and man were estimated by primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labeled nucleotide. Aliquots taken during PCR were analyzed for product formation by two-dimensional densitometry of autoradiograms of separated cDNA on dried agarose gels. The kinetic RT/PCR assay was calibrated for the range of 10(-3) to 10(3) amol of specific mRNA by utilizing mdr1 RNA transcribed from a full-length cDNA as the external standard and aldolase mRNA as the internal standard in order to assess RNA degradation of the specimen. For various cDNA fragments differing between 200 and 600 bp in size, the yield of cDNA during logarithmic growth increased by 1.78 +/- 0.02 per cycle. Hence, the sensitive and reproducible quantitation of transcript is feasible by monitoring RT/PCR kinetics without the need for any competing cRNA or cDNA fragment provided that a high-performance thermal cycler is employed for PCR.