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Comparative Study
. 2021:2300:41-58.
doi: 10.1007/978-1-0716-1386-3_5.

Northern Blot Detection of Tiny RNAs

Jana C Wiegard  1 , M Amri C Schlüter  1 , Olga Y Burenina  2   3 , Elena A Kubareva  3 , Gabriele Klug  4 , Arnold Grünweller  1 , Roland K Hartmann  5
Affiliations
Comparative Study

Northern Blot Detection of Tiny RNAs

Jana C Wiegard et al. Methods Mol Biol. 2021.

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).

Keywords: Digoxigenin; EDC crosslinking; LNA; Native PAGE; Northern blot; UV crosslinking.

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References

    1. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179 - DOI
    1. Pall GS, Codony-Servat C, Byrne J, Ritchie L, Hamilton A (2007) Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res 35:e60 - DOI
    1. Várallyay E, Burgyán J, Havelda Z (2008) MicroRNA detection by northern blotting using locked nucleic acid probes. Nat Protoc 3:190–196 - DOI
    1. Beckmann BM, Grünweller A, Weber MH, Hartmann RK (2010) Northern blot detection of endogenous small RNAs (approximately14 nt) in bacterial total RNA extracts. Nucleic Acids Res 38:e147 - DOI
    1. Beckmann BM, Grünweller A, Hartmann RK (2014) Northern blot detection of endogenous small RNAs. In: Hartmann RK, Bindereif A, Schön A, Westhof E (eds) Handbook of RNA biochemistry, 2nd edn. Wiley-VCH, Weinheim, pp 89–103 - DOI

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