RT-PCR validation of two new AS events identified within the P4HA2 and GSTP1 genes (see text). (Lane 1) A 100-bp ladder. (Lanes 2,4) RT-PCR products obtained with the nonspecific pairs of primers. Because these primers have been designed so that the two members of a pair would anneal the flanking regions on both sides of the corresponding AS event, they make possible the coamplification of the two isoforms produced by the event. Whereas two products with the expected size (a 161-bp and a 334-bp product corresponding, respectively, to the known and new splice variants) are observed with P4HA2 (lane 2), only one product (the expected 265-bp product corresponding to the already known splice variant) is observed with GSTP1 (lane 4). (Lanes 3,5) RT-PCR products obtained with the specific pairs of primers. These primers have been designed so that one member of a pair (nonspecific primer) would anneal flanking regions upstream or downstream of the newly identified AS event, whereas the other member (specific primer) would anneal within the additional sequence introduced by the event (the two new splicing isoforms monitored here are characterized by the insertion of an additional sequence relative to the known isoforms). This design ensures that only the new splicing isoforms can be amplified. For P4HA2 and GSTP1 (lanes 3,5), the expected products corresponding to the new splice variants are observed (a 274-bp and a 134-bp product, respectively). The arrow in lane 5 indicates an additional GSPT1 product at ∼250 bp. Although we didn’t investigate further the identity of this amplification product, we note that its size is consonant with the occurrence of a new AS event corresponding to the retention of the last intron of the GSPT1 gene.
