Protocol abstract
Nature Protocols 3, - 1077 - 1084 (2008)
Published online: 5 June 2008 | doi:10.1038/nprot.2008.67
Subject Categories: Genomics and proteomics | Isolation, purification and separation | Nucleic acid based molecular biology
Improved northern blot method for enhanced detection of small RNA
Gurman S Pall1 & Andrew J Hamilton1
Abstract
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.
- Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, University of Glasgow, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, Scotland, UK.
Correspondence to: Andrew J Hamilton1 e-mail: a.hamilton@clinmed.gla.ac.uk
nature-products
A-Z product listing
- 1-Methylimidazole(Sigma-Aldrich)
- 3MM Whatman chromatography paper(Schleicher & Schuell)
- Acrylamide/bis(Sigma)
- Ammonium persulfate(Sigma)
- Decade RNA markers(Ambion)
- Deionized formamide(Sigma)
