Abstract
Successful detection of very small RNAs (tiny RNAs, ~8–15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5′-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5′-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50–400 nt).
Jana C. Wiegard and M. Amri C. Schlüter are joint first authors.
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (RTG 2355) to RKH and the Russian Foundation for Basic Research (19-04-00791) to OYB and EAK.
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Wiegard, J.C. et al. (2021). Northern Blot Detection of Tiny RNAs. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 2300. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1386-3_5
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DOI: https://doi.org/10.1007/978-1-0716-1386-3_5
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