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Clinical Chemistry 45: 619-624, 1999;
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(Clinical Chemistry. 1999;45:619-624.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

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

Stefan M. Loitsch1, Stefan Kippenberger2, Nurlan Dauletbaev1, Thomas O.F. Wagner1 and Joachim Bargon1,a

1 Department of Internal Medicine, Division of Pulmonary Medicine, and
2 Department of Dermatology, University Hospital Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt, Germany.
a Author for correspondence. Fax 0049-69-63017391; e-mail Bargon{at}t-online.de

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 µg) 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.© 1999 American Association for Clinical Chemistry




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The c-myc x E2F-1/p21 Interactive Gene Expression Index Augments Cytomorphologic Diagnosis of Lung Cancer in Fine-Needle Aspirate Specimens
J. Mol. Diagn., August 1, 2003; 5(3): 176 - 183.
[Abstract] [Full Text] [PDF]


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