QUANTITATIVE REVERSE TRANSCRIPTASE-PCR AMPLIFICATION OF CYTOKINE MESSENGER-RNA IN LIVER-BIOPSY SPECIMENS USING A NONCOMPETITIVE METHOD

Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small sam ples. The aim of this work was to investigate the utility of a non-com petitive RT-PCR which used external standards to quantitate TNF-alpha mRNA in liver biopsy specimens from liver transplant patients. It invo lved removal of aliquots from the PCR reaction at successive cycles, f ollowed by dot-blotting of the samples onto nylon membrane and hybridi zation with a radioactively-labelled internal probe. Phosphorimage ana lysis of the labelled membranes allowed quantitation of the relative a mount of PCR product al successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplif ication. The slopes of these lines showed the efficiency of amplificat ion, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-alpha in two separate PCR amplifications showed low inter-assay variability (r(2) = 0.98). Comparison of two s eparate cDNA syntheses also showed good correlation (r(2) = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that va riation in efficiency of cDNA synthesis is likely to contribute as muc h or more to variability of the analysis as variations in PCR amplific ation.