Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot

Pall, G.S., Codony-Servat, C., Byrne, J., Ritchie, L. and Hamilton, A. (2007) Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Research, 35(8), e60-e60. (doi: 10.1093/nar/gkm112) (PMID:17405769) (PMCID:PMC1885651)

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Publisher's URL: http://dx.doi.org/10.1093/nar/gkm112

Abstract

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40 nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Byrne, Dr Jane and Hamilton, Dr Andrew and Codony-Servat, Dr Carles and Pall, Dr Gurman
Authors: Pall, G.S., Codony-Servat, C., Byrne, J., Ritchie, L., and Hamilton, A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Nucleic Acids Research
ISSN:0305-1048
ISSN (Online):1362-4962
Published Online:07 April 2007

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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