Non-isotopic competitive reverse transcription polymerase chain reaction coupled with high performance liquid...

We describe an application of competitive reverse transcription-polymerase chain reaction (PCR) coupled with HPLC for quantification of beta 2-adrenergic receptor messenger RNA (mRNA) in human atrial tissues removed during cannulation for cardiopulmonary bypass operations. We constructed an internal standard which was reverse transcribed in different concentrations together with constant levels of cellular RNA and subsequently PCR amplified. The competitor RNA shows the same beta 2-adrenergic receptor primer sequences as the cellular mRNA but yields a different-sized product. This allows resolution of the amplified copy DNA (complementary DNA, cDNA) fragments with a specific HPLC column. The concentration of beta 2-adrenergic receptor mRNA is derived from the ratio between the peak intensities corresponding to the amplified competitor and target products. We assessed the imprecision, accuracy and sensitivity of the method. Concentrations of beta 2-adrenergic receptor mRNA of 22.7 +/- 15.2 x 10(6) molecules per micrograms total RNA in patients treated with beta 2-antagonists were not significantly different from control patients showing 16.8 +/- 9.9 x 10(6) beta 2-adrenergic receptor mRNA molecules per microgram total RNA (Mean +/- SD). Competitive reverse transcription PCR is a highly specific, non-radioactive procedure for quantification of beta 2-adrenergic receptor mRNA and simultaneously other gene expression levels of interest in atrial tissue specimens and may therefore be used to advance our understanding of heart muscle disease.