Identification of 3 novel mutations in the CFTR gene, R117P, DD192 and 312111G3A in four French patients
- D Feldmann, A Sardet, E Cougoureux, JL Fontaine, G Tournier, P. Aymard
- Hum Mutat
variation due to tissue inhomogeneity. The CV duplicates (23) for the mRNA concentrations determined in two biopsies from the same placenta (n 5 12) was 38%. In two placentae, the variations between two sets of data (178 and 478 3 10 mol/mg RNA and 273 and 396 3 10 mol/mg RNA) were considerable. When these data were excluded, the CV was reduced to 20%. Based on our results, we recommend examining at least two biopsies from the same tissue and accepting data only if the difference between the samples meets predefined criteria for acceptance. Only a few groups have reported on the imprecision of mRNA analysis using competitive PCR (1–4, 6, 7, 24). Becker-André and Hahlbrock (25) were among the first to describe a competitive PCR assay by adding a standard (competitor) after the reverse transcriptase, allowing mRNA quantification by referring to a housekeeping enzyme mRNA. Two reports using this assay examined three different mRNAs and reported an imprecision between 14% and 27% (1, 3). Because the standard was added after the PCR, this variation reflects the PCR only. In competitive RT-PCR, the standard is added before the reverse transcriptase reaction (26). Ferre (24) developed a competitive RT-PCR analysis for HIV-I with a within-run analytical imprecision of 19%, which is comparable with the within-run imprecision we describe for the EGFr mRNA. The amount of EGFr mRNA in the same samples was determined to be 170 (80–300) 3 10 mol/mg RNA (mean and range, n 5 10). The EGFr protein concentration in the placenta samples was 10.6 (7.4–14.4) 3 10 mol/mg membrane protein (mean and range, n 5 12). We did not find any significant correlation between EGFr mRNA and EGFr protein concentration in the placentae (Pearson r 5 0.30, 2p 5 0.40, n 5 10) (data not shown). The lack of correlation may be explained by a combination of the narrow range covered for the EGFr, the high analytical imprecision for the mRNA analysis, or posttranscriptional regulation.