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

Nature Methods volume 3, pages 833–838 (2006)Cite this article

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).
    Article CAS Google Scholar
  2. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).
    Article CAS Google Scholar
  3. Scacheri, P.C. et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1892–1897 (2004).
    Article CAS Google Scholar
  4. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).
    Article CAS Google Scholar
  5. Doench, J.G. & Sharp, P.A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).
    Article CAS Google Scholar
  6. Echeverri, C.J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nat. Rev. Genet. 7, 373–384 (2006).
    Article CAS Google Scholar
  7. Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).
    Article CAS Google Scholar
  8. Qiu, S., Adema, C.M. & Lane, T.A computational study of off-target effects of RNA interference. Nucleic Acids Res. 33, 1834–1847 (2005).
    Article CAS Google Scholar
  9. Naito, Y. et al. dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference. Nucleic Acids Res. 33, W589–W591 (2005).
    Article CAS Google Scholar
  10. Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004).
    Article CAS Google Scholar
  11. Flockhart, I. et al. FlyRNAi: the Drosophila RNAi screening center database. Nucleic Acids Res. 34, D489–D494 (2006).
    Article CAS Google Scholar
  12. Adams, M.D. et al. The genome sequence of Drosophila melanogaster. Science 287, 2185–2195 (2000).
    Article Google Scholar
  13. Hild, M. et al. An integrated gene annotation and transcriptional profiling approach towards the full gene content of the Drosophila genome. Genome Biol 5, R3 (2003).
    Article CAS Google Scholar
  14. Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005).
    Article CAS Google Scholar
  15. Wharton, K.A., Yedvobnick, B., Finnerty, V.G. & Artavanis-Tsakonas, S. opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster. Cell 40, 55–62 (1985).
    Article CAS Google Scholar
  16. Baeg, G.H., Zhou, R. & Perrimon, N. Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila. Genes Dev. 19, 1861–1870 (2005).
    Article CAS Google Scholar
  17. Dasgupta, R. & Perrimon, N. Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research. Oncogene 23, 8359–8365 (2004).
    Article CAS Google Scholar
  18. Nybakken, K., Vokes, S.A., Lin, T.Y., McMahon, A.P. & Perrimon, N. A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nat. Genet. 37, 1323–1332 (2005).
    Article CAS Google Scholar
  19. Arziman, Z., Horn, T. & Boutros, M. E-RNAi: a web application to design optimized RNAi constructs. Nucleic Acids Res. 33, W582–W588 (2005).
    Article CAS Google Scholar
  20. Gunsalus, K.C., Yueh, W.C., MacMenamin, P. & Piano, F. RNAiDB and PhenoBlast: web tools for genome-wide phenotypic mapping projects. Nucleic Acids Res. 32 (Database issue), D406–D410 (2004).
    Article CAS Google Scholar
  21. Clemens, J.C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97, 6499–6503 (2000).
    Article CAS Google Scholar
  22. Friedman, A. & Perrimon, N. High-throughput approaches to dissecting MAPK signaling pathways. Methods published online 14 July 2006.
  23. Benjamini, Y. & Yekutieli, D. Quantitative trait Loci analysis using the false discovery rate. Genetics 171, 783–790 (2005).
    Article CAS Google Scholar

Download references

Acknowledgements

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.

Author information

Author notes

  1. Serena J Silver
    Present address: Broad Institute at the Massachusetts Institute of Technology, 7 Cambridge Center, Cambridge, Massachusetts, 02142, USA
  2. Meghana M Kulkarni and Matthew Booker: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA
    Meghana M Kulkarni, Matthew Booker, Serena J Silver, Adam Friedman, Norbert Perrimon & Bernard Mathey-Prevot
  2. Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA
    Meghana M Kulkarni, Adam Friedman & Norbert Perrimon
  3. National Center for Behavioral Genomics, Brandeis University, Waltham, 03454, Massachusetts, USA
    Pengyu Hong

Authors

  1. Meghana M Kulkarni
    You can also search for this author inPubMed Google Scholar
  2. Matthew Booker
    You can also search for this author inPubMed Google Scholar
  3. Serena J Silver
    You can also search for this author inPubMed Google Scholar
  4. Adam Friedman
    You can also search for this author inPubMed Google Scholar
  5. Pengyu Hong
    You can also search for this author inPubMed Google Scholar
  6. Norbert Perrimon
    You can also search for this author inPubMed Google Scholar
  7. Bernard Mathey-Prevot
    You can also search for this author inPubMed Google Scholar

Contributions

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 toMeghana M Kulkarni.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

About this article

Cite this article

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). https://doi.org/10.1038/nmeth935

Download citation

This article is cited by