Northern Blot Detection of Tiny RNAs

Methods Mol Biol. 2021:2300:41-58. doi: 10.1007/978-1-0716-1386-3_5.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Northern
  • Cross-Linking Reagents / chemistry*
  • DNA Probes / chemistry
  • DNA Probes / metabolism*
  • Denaturing Gradient Gel Electrophoresis
  • Digoxigenin / chemistry
  • Ethyldimethylaminopropyl Carbodiimide / chemistry*
  • Native Polyacrylamide Gel Electrophoresis
  • RNA / analysis*
  • RNA / chemistry

Substances

  • Cross-Linking Reagents
  • DNA Probes
  • RNA
  • Digoxigenin
  • Ethyldimethylaminopropyl Carbodiimide