Problems associated with the detection of alternative splicing isoforms. (A) Alternative splicing of a single cassette exon (gray box) generates two isoforms differing by one exon. (B) Strategy for specific amplification of the long splicing isoform. The cDNA is amplified using primers specific to the alternative (gray arrow) and the constitutive (black arrow) exons. (C) Strategy for specific amplification of the short splicing isoform. The cDNA is amplified using a boundary-spanning primer (gray arrow) and an opposing primer hybridizing to a constitutive exon (black arrow). (D) Mispriming is often caused by lack of complementarities between the alternative exon and either the 3′ (MP 3′) or 5′ (MP 5′) ends of the BSP on the long splicing isoform. (E) Summary of the RASE pipeline. In this study genes were selected based on the evidence of alternative splicing (NCBI 36.2 database) and the nature of the splicing events.
