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Nucleic Acids Research Advance Access published online on July 15, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn445
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions

Omid R. Faridani1,*, Gerald M. McInerney1,2, Katarina Gradin1 and Liam Good1,3

1Department of Cell and Molecular Biology, Karolinska Institutet, Berzelius väg 35, 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, S-171 77, Stockholm, Sweden and 3Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London. N. Mymms, Herts. AL9 7TA, UK

*To whom correspondence should be addressed: Tel: +46 8 524 87934; Fax: +46 8 337412; Email: omid.faridani{at}ki.se

Received April 29, 2008. Revised June 24, 2008. Accepted June 27, 2008.

Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription–polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells.


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