Figure 2.

Validation of microarray-detected changes in AS and mRNA levels following Pol II elongation inhibition. (A,B) Primer pairs specific for constitutive exons flanking each alternative exon (see Fig. 1A) were used to amplify exon-included and exon-skipped spliced variants, which are indicated by red and yellow circles, respectively. Each RT-PCR analysis was performed in triplicate on total RNA isolated from mock-treated control (“C”) cells, or cells treated with the three different concentrations of DRB (A) or camptothecin (B) (see Fig. 1A). The common color scale represents the percent of transcripts with exon inclusion (%In) and mRNA levels (Trans), as predicted from the microarray data and quantified from the RT-PCR data. Transcript levels on the arcsinh scale were represented as the level in each treatment over the median level for all treatments for the same gene. Numerical values below each panel represent average measurements from the three independent RT-PCR experiments. RT-PCR assays monitoring levels of 5S rRNA and U1 snRNA were used to control for loading and recovery. Additional RT-PCR validation experiments for the same treatments, together with numerical quantification of the data, are shown in Supplemental Figures S2, S3, and S4.