Measuring Poly-Adenosine Tail Length of RNAs by High-Resolution Northern Blotting Coupled with RNase H Cleavage
© 2024. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply..
The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps. The assay provides high specificity, sensitivity, and reproducibility for accurate quantitation using standard molecular biology equipment and reagents. Overall, the high-resolution northern blotting approach offers a cost-effective means of poly(A) RNA analysis that is especially useful for small numbers of transcripts and comparisons between experimental conditions or time points.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2723 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2723(2024) vom: 01., Seite 93-111 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Arvola, René M [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 02.11.2023 Date Revised 04.04.2024 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-3481-3_6 |
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| 520 | |a The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps. The assay provides high specificity, sensitivity, and reproducibility for accurate quantitation using standard molecular biology equipment and reagents. Overall, the high-resolution northern blotting approach offers a cost-effective means of poly(A) RNA analysis that is especially useful for small numbers of transcripts and comparisons between experimental conditions or time points | ||
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