Roadblock-qPCR: A simple and inexpensive strategy for targeted measurements of mRNA stability.

ABSTRACT: The stability of mRNAs is fundamental to determining expression level and dynamics. Nonetheless, current approaches for measuring transcript half-lives (e.g. transcription shutoff) are generally toxic or technically complex. Here we describe an alternative strategy for targeted measurements of endogenous mRNA stability that is simple, inexpensive, and non-toxic. Cells are first metabolically labelled with the nucleoside analog 4-thiouridine (4sU). Extracted mRNA can then be treated with the thiol-reactive compound N-ethylmaleimide. This compound modifies 4sU nucleotides and sterically interferes with reverse transcription of 4sU-containing transcripts, disrupting their conversion into cDNA. The decay rate of non-4sU-containing pre-existing mRNA can then be monitored by quantitative PCR (qPCR). Importantly, this approach avoids the biochemical isolation of 4sU-labelled transcripts and/or RNA-seq analysis required for other metabolic-labelling strategies. In summary, our method combines the simplicity of "transcription shutoff" strategies with the accuracy of metabolic-labelling strategies for measurements of mRNA stability across a wide range of half-lives.

SUBMITTER: Watson M 

PROVIDER: S-EPMC7901842 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature