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. 2022:2537:63-79.
doi: 10.1007/978-1-0716-2521-7_5.

Quantitative Measurement of Alternatively Spliced RNA Isoform Levels

Zhelin Jeff Li  1 , Sika Zheng  2
Affiliations

Quantitative Measurement of Alternatively Spliced RNA Isoform Levels

Zhelin Jeff Li et al. Methods Mol Biol. 2022.

Abstract

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.

Keywords: AS-NMD; Alternative splicing; Exon; Intron; Isoform specific; Junction primers; Quantitation; RT-qPCR; Reporter.

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