Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays.
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 > or = 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.
Kulkarni
Meghana M
MM
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. mkulkarni@receptor.med.harvard.edu
Booker
Matthew
M
Silver
Serena J
SJ
Friedman
Adam
A
Hong
Pengyu
P
Perrimon
Norbert
N
Mathey-Prevot
Bernard
B
D000818:Animals; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics; D005189:False Positive Reactions; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D012680:Sensitivity and Specificity
D012330:RNA, Double-Stranded