In vitro splicing of Itga2 pre-mRNA. (A) Itga2 pre-mRNA containing CA6 or CA21 was incubated under splicing conditions with a nuclear extract from B16F10 cells for 0, 30, or 90 minutes. The spliced RNA products were then separated on a 6% denaturing polyacrylamide gel, and RNA bands were detected by staining with SYBR gold. The positions of size standards representing 200 or 300 nucleotides is indicated to the left of the gel. The position of pre-mRNA (black arrowhead) and spliced products (arrows A and B) are indicated. Product B represents the correctly spliced exon 1-exon 2 product. The yield of product B is significantly greater in the presence of CA21, relative to CA6. (B) In vitro splicing efficiency. The relative amounts of the correctly spliced exon1-exon2 products (band B in panel A) were determined by optical densitometry using image J.38 Optical densities divided by unit area (square pixels) for 3 independent assays were obtained, and the mean ± SD is depicted. The mean density obtained in the case of the CA21 probe (0.23) is significantly higher than that obtained for the CA6 probe (0.15).
