Competitive reverse transcription polymerase chain reaction for quantifying pre-mRNA and mRNA of major acute phase...

Competitive reverse transcription polymerase chain reaction (RT-PCR) is an increasingly used method for quantifying RNA. The technique involves co-amplification from test RNA with an internal standard using common primers in a single reaction. The standard competes for primers and enzyme and it is therefore referred to as a competitor. A RT-PCR polycompetitor for use in quantifying acute phase serum amyloid A, constitutive serum amyloid A, serum amyloid P component, C-reactive protein, apolipoprotein A1, apolipoprotein A2, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNAs has been generated and used in quantitative PCR. The polycompetitor was synthesised from ten oligonucleotides of 77-90 bases using primer extension and contains sequences which permit amplification using priming sites that are present in both hnRNA (pre-mRNA) and mRNA. The polycompetitor was cloned into the expression vector pSP64(polyA) and a polycompetitor transcript with a poly(A)-tail sequence at the 3'-end was generated by in vitro transcription. The poly(A)-tail sequence allows the option of performing reverse transcription using oligo(dT) in addition to directed reverse transcription using the specific 3'-reverse PCR primers. cDNA products generated from amplification of the internal polycompetitor standard and endogenous RNA species were separated by capillary electrophoresis and quantified by UV absorbance at 254 nm. Reproducibility was determined to be very high when starting ratios of internal standard and target mRNA are at an approximate equivalence point. Relative standard deviations were less than 5% between independent RT-PCR reactions with the same sample mix of internal standard and total RNA. Applying the method to total RNA samples harvested at various timepoints following cytokine induction of acute phase mRNAs in endothelial cells demonstrated that quantitative readout from the RT-PCR method correlates well with that obtained from Northern-blotting and is at least 100-fold more sensitive. This method will be useful for studying regulation of acute phase proteins in in vitro tissue culture experiments and may also be applied to clinical tissue samples from patients with inflammatory diseases.