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.
Pall
Gurman S
GS
Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, University of Glasgow, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, Scotland, UK.
Hamilton
Andrew J
AJ
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