Validation of alternative mRNA isoforms in polyribosomal and cytosolic subcellular fractions by RT-PCR. (A) Correlation of endpoint RT-PCR (in triplicate with standard error) determined ΔΨ with average MISO estimated ΔΨ. Linear regression plotted as a solid line with 95% confidence interval (shaded region). Significance P-value derived from t-distribution test using Pearson's product-moment correlation coefficient. (B) Representative agarose gels of endpoint RT-PCR for two exemplar genes, HNRNPR and KARS. C1 and C2 refer to the cytoplasmic derived replicates, whereas P1 and P2 refer to the polyribosome-bound replicates. (C,D) Sashimi plots depict RPKM coverage of RNA-seq reads used to estimate Ψ values for HNRNPR and KARS, respectively. Junction reads are annotated as connecting lines.
