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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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.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| 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: | 1754-2189 |
| ISSN (Online): | 1750-2799 |
| Published Online: | 05 June 2008 |
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