Non-Isotopic Competitive Reverse Transcription Polymerase Chain Reaction Coupled with High Performance Liquid Chromatography to Measure β ₂ -Receptor Messenger RNA in the Human Heart

Abstract

Summary:We describe an application of competitive reverse transcription-polymerase chain reaction (PCR) coupled with HPLC for quantification of 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 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 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 2 -adrenergic receptor mRNA of 22.7 ± 15.2 Χ 10 6 molecules pe^g total RNA in patients treated with 2 -antagonists were not significantly different from control patients showing 16.8 ± 9.9 Χ 10 6 2 -adrenergic receptor mRNA molecules per μg total RNA (Mean ± SD). Competitive reverse transcription PCR is a highly specific, non-radioactive procedure for quantification of 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.

Polyclonal anti-histamine H 2 receptor antibodies detect differential expression of H 2 receptor protein in primary vascular cell types

“…Competitive RT-PCR is a very sensitive method that allows for the direct quantification of the amount of specific mRNA molecules per µg total RNA permitting a direct comparison of H 2 receptor mRNA concentrations between different cell types or tissues [30]. The close correlation of H 2 receptor mRNA concentration and H 2 receptor protein levels in the primary vascular cell types indicates the efficacy and usefulness of the newly generated polyclonal H 2 receptor antibodies for the determination and quantification of H 2 receptor protein expression.…”

Quantification of Low Abundance Natriuretic Peptide Receptor mRNA in Rat Tissues

“…The intersection between the regression curve of standard and sample PCR products represents the equimolar ratio of molecules of sample and standard RNA initially present in the assay, if both PCR products are equal in length. Since the two PCR products are different in length, the initially calculated equivalence point had to be multiplied by a factor which is calculated by division of the standard PCR product length and the target PCR product, length in base pairs (13). The in vitro transcribed singlestranded RNA consists of 214 ribonucleotides and the molecular weight was calculated to be 72974 daltons.…”