Figure 2

Analysis of delta3-transcript expression. a: Fragment analysis of Onestep RT-PCR products spanning exon 2 to 6. Semi-quantitative fragment analysis of Onestep RT-PCR products revealed the presence of the wild type transcripts at 477 bp and the delta3 alternative transcripts at 231 bp, as shown in the electrophoretograms of a control sample (wild type) and samples with the c.68-7T>A and c.316+3delA mutations. The percentages of delta3-transcript were, respectively: 6%, 22% and 61% for the sample illustrated. Left upper panels: agarose gel images of the same products after 33 cycles of PCR; the left wells contain the control, the right wells the samples with the respective mutation. b: Pyrosequencing histogram from the pyrogram for the c.-26A>G polymorphism with the primer set Ex1-2 F and Ex2-Ex3/4 R (only the full-length transcript). The sequence is C/T TTGGTAAATA. The proportion of C and T is directly computed by the pyrosequencing software. c: Pyrosequencing histogram from the pyrogram for the competitive QPCR using primer set exon 2/exon 3 and exon 2/exon 4 for the full-length transcript and delta3-transcripts, respectively. The sequence of the exon 2/3 transcript is CAGATTT and the sequence of the exon 2/4 transcript is CAGGAA. The proportion of GA (full-length transcript) and GGA (delta3-transcript) is directly computed on the pyrosequencing software.