Improved northern blot method for enhanced detection of small RNA

Nat Protoc. 2008;3(6):1077-84. doi: 10.1038/nprot.2008.67.


This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.

MeSH terms

  • Animals
  • Blotting, Northern / methods*
  • Cross-Linking Reagents
  • Embryonic Stem Cells / chemistry
  • Mice
  • MicroRNAs / genetics
  • MicroRNAs / isolation & purification*
  • Phosphorus Radioisotopes
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / isolation & purification*


  • Cross-Linking Reagents
  • MicroRNAs
  • Phosphorus Radioisotopes
  • RNA, Small Interfering