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Reverse transcription-competitive multiplex PCR improves quantification of mRNA in clinical samples--application to
S M Loitsch1, S Kippenberger, N Dauletbaev
1Department of Internal Medicine, Division of Pulmonary Medicine, and.
Background:
To monitor gene therapy, we wished to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. We developed a PCR-based method to measure CFTR mRNA in clinical samples.
Methods:
Expression was determined by reverse transcription-competitive multiplex PCR (RCMP) for CFTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, and for serial dilutions of two internal cDNA standards consisting of CFTR and GAPDH mutants containing short deletions. The RCMP used 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 microgram) 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 high variability of CFTR expression in non-cystic fibrosis donors.
Conclusion:
This method is precise and reproducible and advantageous for use with limited amounts of tissue, such as from biopsies or from nasal or bronchial brushings.