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cge12154-sup-0001-FigureS1.tifTIFF image203KFig S1. RT-PCR analysis in patients' fibroblasts. (a) Full-length cDNA was amplified with a set of primers encompassing exons 1–7. The analysis revealed a shorter DM20 product of 745-bp in size, corresponding to an anomalous isoform with the entire skipping of exon 4 in the two probands (lanes 2 and 3) compared to the two normal, nonaffected, controls (lanes 1 and 4) showing the expected product (914-bp). Note that the PLP product (1019-bp) is not visible in any of the samples likely due to the low amount of PLP transcribed in fibroblasts. (b) To selectively identify the PLP product, two consecutive nested PCRs were performed on full-length cDNA product: (i) the first nested PCR, performed by a set of primers lying on exons 1 and 3B, amplified the expected PLP product (558-bp) in both the patients (lanes 2 and 3) and the controls (lanes 1 and 4); (ii) the second nested PCR, performed with a set of primers lying on exons 3B and 7, revealed the expected product (565-bp) in normal controls (lanes 1 and 4), while a shorter product of 396-bp, corresponding to the entire skipping of exon 4, in the patients (lanes 2 and 3). Note that the primers lying on exon 3B are PLP sequence specific (details in Fig. 2). M: ΦX DNA HaeIII digested molecular weight marker; C: no template control.
cge12154-sup-0002-FigureS2.tifTIFF image100KFig S2. PLP1 exon 4-specific RT-PCR. The analysis, performed on RNA samples (extracted from fibroblasts), PCR amplified by a set of primers, lying on exons 2 and 4, showed the expected DM20 isoform (348 bp) in the normal, nonaffected, controls (lanes 3 and 4), while no product was present in the two probands (lanes 1 and 2). Note that the other expected product of 453 bp in size (PLP isoform) is not visible in any of the samples likely due to the low amount of PLP isoform transcribed in the fibroblasts. M: ΦX DNA HaeIII digested molecular weight marker; C: no template control.

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