Access

Article

Nature Methods 3, 833–838 (1 October 2006) | doi:10.1038/nmeth935

Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays

Meghana M Kulkarni , Matthew Booker , Serena J Silver , Adam Friedman , Pengyu Hong , Norbert Perrimon & Bernard Mathey-Prevot

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 |[ge]|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.