Abstract
Successful detection of very small RNAs (tiny RNA, ~14 nt in length) by Northern blotting is dependent on improved Northern blot protocols that combine chemical crosslinking of RNA with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to positively charged membranes, the use of native polyacrylamide gels, and the development of highly sensitive and specific probes modified with locked nucleic acids (LNA). In this protocol, 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.
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Acknowledgment
This work was supported by the Deutsche Forschungsgemeinschaft (GK 1384) to R.K.H. and G.K. and the Russian Foundation for Basic Research (14-04-91336) to O.Y.B. and E.A.K.
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Damm, K. et al. (2015). Improved Northern Blot Detection of Small RNAs Using EDC Crosslinking and DNA/LNA Probes. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 1296. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2547-6_5
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DOI: https://doi.org/10.1007/978-1-4939-2547-6_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2546-9
Online ISBN: 978-1-4939-2547-6
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