Skip to main content
SpringerLink
Log in

In Vitro RISC Cleavage Assay

  • Protocol
  • First Online:
Argonaute Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 725))

Abstract

RNA interference (RNAi) is a widely used tool for the analysis of gene expression. In this process, short-interfering RNAs (siRNAs) guide the RNA-induced silencing complex (RISC) to complementary target RNA molecules, which are sequence-specifically cleaved by the RISC. In vitro cleavage assays have proved to be powerful tools for the characterization of the RNAi pathway in many different organisms. Therefore, this chapter provides a detailed protocol for in vitro RISC assays.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391, 806–811.

    Google Scholar 

  2. Kim, D. H., and Rossi, J. J. (2007) Strategies for silencing human disease using RNA interference, Nat Rev Genet 8, 173–184.

    Google Scholar 

  3. Meister, G., and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA, Nature 431, 343–349.

    Google Scholar 

  4. Siomi, H., and Siomi, M. C. (2009) On the road to reading the RNA-interference code, Nature 457, 396–404.

    Google Scholar 

  5. Khvorova, A., Reynolds, A., and Jayasena, S. D. (2003) Functional siRNAs and miRNAs exhibit strand bias, Cell 115, 209–216.

    Google Scholar 

  6. Schwarz, D. S., Hutvágner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P. D. (2003) Asymmetry in the assembly of the RNAi enzyme complex, Cell 115, 199–208.

    Article  PubMed  CAS  Google Scholar 

  7. Peters, L., and Meister, G. (2007) Argonaute proteins: mediators of RNA silencing, Mol Cell 26, 611–623.

    Google Scholar 

  8. Hutvagner, G., and Simard, M. J. (2007) Argonaute proteins: key players in RNA silencing, Nat Rev Mol Cell Biol 9, 22–32.

    Article  Google Scholar 

  9. Jinek, M., and Doudna, J. A. (2009) A three-dimensional view of the molecular machinery of RNA interference, Nature 457, 405–412.

    Google Scholar 

  10. Ma, J. B., Yuan, Y. R., Meister, G., Pei, Y., Tuschl, T., and Patel, D. J. (2005) Structural basis for 5’-end-specific recognition of guide RNA by the A. fulgidus Piwi protein, Nature 434, 666–670.

    Google Scholar 

  11. Parker, J. S., Roe, S. M., and Barford, D. (2005) Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex, Nature 434, 663–666.

    Article  PubMed  CAS  Google Scholar 

  12. Wang, Y., Juranek, S., Li, H., Sheng, G., Wardle, G. S., Tuschl, T., and Patel, D. J. (2009) Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes, Nature 461, 754–761.

    Google Scholar 

  13. Song, J. J., Smith, S. K., Hannon, G. J., and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity, Science 305, 1434–1437.

    Google Scholar 

  14. Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and Tuschl, T. (2004) Human Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs, Mol Cell 15, 185–197.

    Google Scholar 

  15. Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J., Hammond, S. M., Joshua-Tor, L., and Hannon, G. J. (2004) Argonaute2 is the catalytic engine of mammalian RNAi, Science 305, 1437–1441.

    Google Scholar 

  16. Martinez, J., Patkaniowska, A., Urlaub, H., Lührmann, R., and Tuschl, T. (2002) Single-stranded antisense siRNAs guide target RNA cleavage in RNAi, Cell 110, 563–574.

    Google Scholar 

  17. Rudel, S., Flatley, A., Weinmann, L., Kremmer, E., and Meister, G. (2008) A multifunctional human Argonaute2-specific monoclonal antibody, Rna 14, 1244–1253.

    Google Scholar 

  18. Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) RNA interference is mediated by 21 and 22 nt RNAs, Genes Dev 15, 188–200.

    Google Scholar 

Download references

Acknowledgments

Our research is supported by the Max-Planck Society, Regensburg University and by grants from the Deutsche Forschungsgemeinschaft (DFG), the European Union (FP6, ERC), the German ministry for education and science (BMBF), and the Bavarian Genome research network (BayGene).

Author information

Authors and Affiliations

  1. Center for Integrated Protein Science Munich (CIPSM), Laboratory for RNA Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany

    Gunter Meister

  2. University of Regensburg, Regensburg, Germany

    Gunter Meister

Authors
  1. Julia Stoehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gunter Meister
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gunter Meister .

Editor information

Editors and Affiliations

  1. , Department of Cell Biology, University of Alberta, Edmonton, T6G 2H7, Alberta, Canada

    Tom C. Hobman

  2. Goodman Cancer Research Centre, Department of Biochemistry, McGill University, Avenue des Pins Ouest 1160, Montreal, H3A 1A3, Québec, Canada

    Thomas F. Duchaine

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Stoehr, J., Meister, G. (2011). In Vitro RISC Cleavage Assay. In: Hobman, T., Duchaine, T. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 725. Humana Press. https://doi.org/10.1007/978-1-61779-046-1_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-046-1_6

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-045-4

  • Online ISBN: 978-1-61779-046-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation