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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 ≥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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Figure 1: Signature difference across 30 screens shown by pairs of dsRNAs that differ in their number of predicted off-target sequences but target the same gene.
Figure 2: Off-target sequences in the DRSC dsRNAs collection and their effect on hit rate frequency.
Figure 3: Predicted off-target sequences in dsRNAs lead to false positives in an ERK activation RNAi screen.
Figure 4: Comparison of fold change expression differences and knockdown efficiency after treatment with pairs of dsRNAs directed against PP2A-B′.

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We thank G.-H. Baeg, S. Cherry, R. DasGupta, J. Phillips, K. Nybakken and R. Zhou for helpful discussions, and K. Nybakken for his gift of the PCR templates for the C1, C2 and C3 dsRNAs. A.F. is a recipient of the Medical Scientist Training Program (MSTP) grant. N.P. is an investigator of the Howard Hughes Medical Institute. Work at the DRSC is supported by grant R01 GM067761 from the US National Institute of General Medical Sciences.

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Authors and Affiliations



M.M.K., microarray design and analysis, RT-qPCR analysis of off-target effects; M.B., S.J.S. and P.H., data analysis and statistical evaluation; A.F., validation of dsRNAs.

Corresponding author

Correspondence to Meghana M Kulkarni.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Microarray analysis of off-target effects. (PDF 517 kb)

Supplementary Fig. 2

Flow-chart and recommendations for assessing the specificity of dsRNAs in genome-wide-screens. (PDF 90 kb)

Supplementary Table 1

GO functional categories specifically enriched in dsRNAs that scored as hits in 5 or more screens. (XLS 94 kb)

Supplementary Table 2

Analyzing the dependence between gene functions and the number of predicted off-targets. (XLS 57 kb)

Supplementary Table 3

Amplicon ID for dsRNAs used in Figure 3. (PDF 34 kb)

Supplementary Table 4

Phenotype of multiple amplicons targeting seven genes in ERK activation assay. (PDF 46 kb)

Supplementary Table 5

Assay reproducibility and discovery rate for known components of the Wg, Hh and JAK/STAT pathways measured in 3 genome-wide RNAi screens. (PDF 48 kb)

Supplementary Methods (PDF 60 kb)

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Kulkarni, M., Booker, M., Silver, S. et al. Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat Methods 3, 833–838 (2006).

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