Nature Methods 2, 967 - 973 (2005)
Published online: 18 November 2005; | doi:10.1038/nmeth812
High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasionHui-Fen Zhao1, Denis L'Abbé1, Normand Jolicoeur1, Meiqun Wu1, Zhen Li1, Zhenbao Yu1
& Shi-Hsiang Shen1, 21
Health Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Québec, Canada, H4P 2R2. 2
Department of Medicine, McGill University, Montréal, Québec, Canada, H3G 1A4.
Correspondence should be addressed to Hui-Fen Zhao hui-fen.zhao@cnrc-nrc.gc.ca or Shi-Hsiang Shen shi.shen@cnrc-nrc.gc.ca Use of RNA interference (RNAi) as a reverse genetics tool for silencing genes in mammalian cells is achieved by in vitro transfection of small interfering RNAs (siRNAs). For a target gene, several siRNAs must be designed according to the empirical rules. We demonstrated that functional short hairpin RNAs (shRNAs) could be synthesized in Escherichia coli and delivered directly via bacterial invasion to the near entirety of a mammalian cell population to trigger RNAi. Furthermore, using a luciferase–target gene transcript, we identified effective shRNAs and siRNAs from RNAi libraries delivered conveniently through bacterial invasion in 96-well plates without need for preparation, purification and transfection of shRNAs. Notably, several of the most highly effective shRNAs and siRNAs identified do not fit the empirical rules commonly used for siRNA design, suggesting that this approach is a powerful tool for RNAi research, and could be used complementarily to the empirical rules for RNAi applications.
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