Reverse transcription-competitive multiplex PCR improves quantification ofmRNA in clinical samples - Application to the low abundance CFTR mRNA

Background: To monitor gene therapy, we wished to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. We developed a PCR-based m ethod to measure CFTR mRNA in clinical samples. Methods: Expression was determined by reverse transcription-competitive mul tiplex PCR (RCMP) for CFTR and glyceraldehyde-3-phosphate dehydrogenase (GA PDH) transcripts, and for serial dilutions of two internal cDNA standards c onsisting of CFTR and GAPDH mutants containing short deletions. The RCMP us ed simultaneous amplification of the gene of interest with a reporter gene in one reaction tube. The expression of CFTR was calculated with reference to the amount of GAPDH to correct for variations in initial RNA loading. Results: Amplification of cDNAs derived from different amounts of RNA (1-4 mu g) gave similar GAPDH/CFTR ratios, with a coefficient of variation (CV) below 7.5%. RCMP was applied on nasal and bronchial brushings and shows a h igh variability of CFTR expression in non-cystic fibrosis donors. Conclusion: This method is precise and reproducible and advantageous for us e with limited amounts of tissue, such as from biopsies or from nasal or br onchial brushings. (C) 1999 American Association for Clinical Chemistry.