Northern Blot Detection of Tiny RNAs

Jana C. Wiegard, M. Amri C. Schlüter, Olga Y. Burenina, Elena A. Kubareva, Gabriele Klug, Arnold Grünweller, Roland K. Hartmann

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Citation (Scopus)

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).

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages41-58
Number of pages18
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2300
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Digoxigenin
  • EDC crosslinking
  • LNA
  • Native PAGE
  • Northern blot
  • UV crosslinking

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