Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 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.

PubMed Disclaimer

References

    1. Lee Y, Rio DC (2015) Mechanisms and regulation of alternative pre-mRNA splicing. Annu Rev Biochem 84:291–323 - DOI
    1. Scotti MM, Swanson MS (2016) RNA mis-splicing in disease. Nat Rev Genet 17:19–32 - DOI
    1. Furlanis E, Scheiffele P (2018) Regulation of neuronal differentiation, function, and plasticity by alternative splicing. Annu Rev Cell Dev Biol 34:451–469 - DOI
    1. Manning KS, Cooper TA (2017) The roles of RNA processing in translating genotype to phenotype. Nat Rev Mol Cell Biol 18:102–114 - DOI
    1. Vuong CK, Black DL, Zheng S (2016) The neurogenetics of alternative splicing. Nat Rev Neurosci 17:265–281 - DOI

Publication types

LinkOut - more resources