Improved northern blot method for enhanced detection of small RNA

Pall, G. S. and Hamilton, A. J. (2008) Improved northern blot method for enhanced detection of small RNA. Nature Protocols, 3(6), pp. 1077-1084. (doi: 10.1038/nprot.2008.67) (PMID:18536652)

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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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Hamilton, Dr Andrew
Authors: Pall, G. S., and Hamilton, A. J.
College/School:College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing > Clinical Specialities
Journal Name:Nature Protocols
Publisher:Nature Publishing
ISSN (Online):1750-2799
Published Online:05 June 2008

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
365401Improving cross-kingdom techniques for siRNA detection and their specific application to x-chromosome inactivationAndrew HamiltonBiotechnology and Biological Sciences Research Council (BBSRC)BBS/B/07306Clinical Specialties