Improved northern blot method for enhanced detection of small RNA.
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.
Gurman S
GS
Pall
Andrew J
AJ
Hamilton
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D003432:Cross-Linking Reagents; D053595:Embryonic Stem Cells / Q000737:chemistry; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D034741:RNA, Small Interfering / Q000235:genetics / Q000302:isolation & purification*
D003432:Cross-Linking Reagents; D035683:MicroRNAs; D010761:Phosphorus Radioisotopes; D034741:RNA, Small Interfering