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Nucleic Acids Research 2005 33(18):e151; doi:10.1093/nar/gni144
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Published online 7 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification

Ming Jiang, Andrey A. Arzumanov1, Michael J. Gait1 and Jo Milner*

YCR P53 Research Laboratory, Department of Biology, University of York York YO10 5DD, UK 1Medical Research Council, Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, UK

*To whom correspondence should be addressed. Tel: +44 1904 328620; Fax: +44 1904 328622; Email: ajm24{at}york.ac.uk

Received June 3, 2005. Revised August 3, 2005. Accepted September 13, 2005.

RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3' end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed ‘crook’ siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics.


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