Quantitative determination of gene expression in human lymphocytes assessed by reverse transcription–polymerase chain reaction coupled to high-performance liquid chromatography

Abstract

Gene expression in human lymphocytes was assessed using reverse transcription and polymerase chain reaction amplification followed by ion-pair reversed-phase chromatography analysis. Competitive PCR was used to quantitate the desired cDNAs with a polivalent competitor adaptable to multiple novel mRNAs estimations with minor changes. Accuracy was 11.27±11.87% ( n=7), as determined using standards. The coefficients of variation of the assessment of human OK12b were 7% ( n=6), 7.68 attmol/μg of total RNA, and 21% ( n=6), 0.93 attmol/μg of total RNA. Sample-to-sample variation in the reverse transcription and in the quantity and quality of RNA was attenuated by normalising results to beta-actin mRNA expression. The correlation between the OK12b/β-actin ratio and competitive assessments of OK12b was 0.984, n=6. The correlation between HPLC results and an independent method based on radionuclide uptake by the product, detected by electrophoretic separation, was 0.848, n=10.