Quantitative Measurement of Alternatively Spliced RNA Isoform Levels.

Conventional approaches to quantify alternative splicing are exon-centric and derive a ratio based on relative levels of the isoforms (or isoform groups) that include versus exclude a particular alternative RNA segment. The ratio measurement to study alternative splicing regulation can be confounded when alternative isoforms undergo differential RNA decay, for example, nonsense-mediated mRNA decay (NMD). Isoform-centric quantification is more informative for functional studies of alternative splicing, but challenges remain in distinguishing specific isoforms. Here, we provide a practical guide on addressing the specificity of isoform quantification and describe a simple sensitive method. Quantitative measurement of alternatively spliced RNA isoforms can be used to differentiate splicing regulation from transcriptional control and isoform-specific RNA decay regulation.