Nature Methods
- 3, 833 - 838 (2006)
Published online: 21 September 2006; | doi:10.1038/nmeth935
Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assaysMeghana M Kulkarni1, 2, 5, Matthew Booker1, 5, Serena J Silver1, 4, Adam Friedman1, 2, Pengyu Hong3, Norbert Perrimon1, 2 & Bernard Mathey-Prevot11
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 2
Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 3
National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts 03454, USA. 4
Present address: Broad Institute at the Massachusetts Institute of Technology, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. 5
These authors contributed equally to this work.
Correspondence should be addressed to Meghana M Kulkarni mkulkarni@receptor.med.harvard.edu To evaluate the specificity of long dsRNAs used in high-throughput RNA interference (RNAi) screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Notably, our analysis predicts that dsRNAs containing  19-nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to efficient knockdown of a cross-hybridizing transcript, raising a cautionary note about interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high-quality information from RNAi high-throughput screens.
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