Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability
- Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06510, USA
- Corresponding author: carson.thoreen{at}yale.edu
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 nontoxic. Cells are first metabolically labeled 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 preexisting mRNA can then be monitored by quantitative PCR (qPCR). Importantly, this approach avoids the biochemical isolation of 4sU-labeled transcripts and/or RNA-seq analysis required for other metabolic-labeling strategies. In summary, our method combines the simplicity of “transcription shutoff” strategies with the accuracy of metabolic-labeling strategies for measurements of mRNA stability across a wide range of half-lives.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.076885.120.
- Received June 17, 2020.
- Accepted November 28, 2020.
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