Reverse Transcription-Competitive Multiplex PCR Improves Quantification of mRNA in Clinical Samples—Application to the Low Abundance CFTR mRNA

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. Expression was determined by reverse transcription-competitive multiplex PCR (RCMP) for CFTR and glyceraldehyde-3-...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 1999-05, Vol.45 (5), p.619-624
Main Authors: Loitsch, Stefan M, Kippenberger, Stefan, Dauletbaev, Nurlan, Wagner, Thomas O.F, Bargon, Joachim
Format: Article
Language:English
Subjects:
Biological and medical sciences
Bronchi - chemistry
Bronchi - cytology
Cells, Cultured
Cystic Fibrosis Transmembrane Conductance Regulator - analysis
Cystic Fibrosis Transmembrane Conductance Regulator - genetics
Electrophoresis, Agar Gel
Glyceraldehyde-3-Phosphate Dehydrogenases - analysis
Glyceraldehyde-3-Phosphate Dehydrogenases - genetics
Humans
Investigative techniques, diagnostic techniques (general aspects)
Medical sciences
Mutation
Nose - chemistry
Nose - cytology
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Reproducibility of Results
Respiratory system
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger - analysis
Sensitivity and Specificity
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Summary: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. 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. 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. 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.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/45.5.619