Nucleic Acids Research Advance Access originally published online on April 17, 2008
Nucleic Acids Research 2008 36(9):e53; doi:10.1093/nar/gkn190
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Nucleic Acids Research, 2008, Vol. 36, No. 9 e53
© 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 |
Analysis of siRNA specificity on targets with double-nucleotide mismatches
1Department of Molecular Medicine and Surgery, Karolinska Institutet, CMM L8:01, 171 76 Stockholm, Sweden, 2Institute of Molecular Medicine, Peking University, Beijing 100871, China and 3Neuroscience Discovery, Scripps Florida, 5353 Parkside Drive RF2, Jupiter, FL 33458, USA
*To whom correspondence should be addressed. Tel: +46 733 152665; Fax +86 10 62769862; Email: dahlgren.cecilia{at}gmail.com
Correspondence may also be addressed to Zicai Liang. Tel: +86 10 62769862; Fax: +86 10 62769862; Email: liangz{at}pku.edu.cn or zicai.liang{at}ki.se
Received December 6, 2007. Revised March 29, 2008. Accepted April 1, 2008.
Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that 
35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.
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