NONISOTOPIC COMPETITIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION COUPLED WITH HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TO MEASURE BETA(2)-RECEPTOR MESSENGER-RNA IN THE HUMAN HEART

We describe an application of competitive reverse transcription-polyme rase chain reaction (PCR) coupled with HPLC for quantification of beta (2)-adrenergic receptor messenger RNA (mRNA) in human atrial tissues r emoved during cannulation for cardiopulmonary bypass operations. We co nstructed an internal standard which was reverse transcribed in differ ent concentrations together with constant levels of cellular RNA and s ubsequently PCR amplified. The competitor RNA shows the same beta(2)-a drenergic 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. T he concentration of beta(2)-adrenergic receptor mRNA is derived from t he ratio between the peak intensities corresponding to the amplified c ompetitor and target products. We assessed the imprecision, accuracy a nd sensitivity of the method. Concentrations of beta(2)-adrenergic rec eptor mRNA of 22.7 +/- 15.2 x 10(6) molecules per mu g total RNA in pa tients treated with beta(2)-antagonists were not significantly differe nt from control patients showing 16.8 +/- 9.9 x 10(6) beta(2)-adrenerg ic receptor mRNA molecules per mu g total RNA (Mean +/- SD). Competiti ve reverse transcription PCR is a highly specific, non-radioactive pro cedure for quantification of beta(2)-adrenergic receptor mRNA and simu ltaneously other gene expression levels of interest in atrial tissue s pecimens and may therefore be used to advance our understanding of hea rt muscle disease.