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

Gene expression in human lymphocytes was assessed using reverse transcripti on and polymerase chain reaction amplification followed by ion-pair reverse d-phase chromatography analysis. Competitive PCR was used to quantitate the desired cDNAs with a polivalent competitor adaptable to multiple novel mRN As 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/mug of total RNA, and 21% (n=6), 0.93 attmol/mug of total RNA. Sample-to-sample variation in the reverse tr anscription and in the quantity and quality of RNA was attenuated by normal ising results to beta-actin mRNA expression, The correlation between the OK 12b/beta -actin ratio and competitive assessments of OK12b was 0.984, n=6. The correlation between HPLC results and an independent method based on rad ionuclide uptake by the product, detected by electrophoretic separation, wa s 0.848, n=10. (C) 2001 Elsevier Science B.V. All rights reserved.