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Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability
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 endo...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901842/ https://www.ncbi.nlm.nih.gov/pubmed/33288682 http://dx.doi.org/10.1261/rna.076885.120 |
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author | Watson, Maegan J. Park, Yeonwoo Thoreen, Carson C. |
author_facet | Watson, Maegan J. Park, Yeonwoo Thoreen, Carson C. |
author_sort | Watson, Maegan J. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7901842 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-79018422022-03-01 Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability Watson, Maegan J. Park, Yeonwoo Thoreen, Carson C. RNA Method 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. Cold Spring Harbor Laboratory Press 2021-03 /pmc/articles/PMC7901842/ /pubmed/33288682 http://dx.doi.org/10.1261/rna.076885.120 Text en © 2021 Watson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Method Watson, Maegan J. Park, Yeonwoo Thoreen, Carson C. Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability |
title | Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability |
title_full | Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability |
title_fullStr | Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability |
title_full_unstemmed | Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability |
title_short | Roadblock-qPCR: a simple and inexpensive strategy for targeted measurements of mRNA stability |
title_sort | roadblock-qpcr: a simple and inexpensive strategy for targeted measurements of mrna stability |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901842/ https://www.ncbi.nlm.nih.gov/pubmed/33288682 http://dx.doi.org/10.1261/rna.076885.120 |
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