Skip to main content

Strategies for the Design of Random siRNA Libraries and the Selection of anti-GFP siRNAs

  • Protocol
RNA Silencing

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 309))

Abstract

Various methods have been devised to elucidate gene function in a highthroughput format. With the potential to silence any gene once the sequence is known, small, interfering RNAs (siRNAs) have been considered ideal for functional analysis and gene target validation (13). Furthermore, the technology has unprecedented target flexibility. By performing RNAi on a large scale using siRNA libraries, this reverse-genetic method can essentially be used as a forward-genetic screening tool. In this respect, genomewide siRNA screens in Caenorhabditis elegans using libraries of in vitro-transcribed long doublestranded RNAs (dsRNAs) have been useful in gene discovery and functional annotation in various processes, including embryonic development (4,5).

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Elbashir, S. M., Harborth, M. J., Ledecknel, W., Yalcin, A., Weber, K, and Tuchl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.

    Article  PubMed  CAS  Google Scholar 

  2. Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.

    Article  PubMed  CAS  Google Scholar 

  3. Sioud, M. (2004) Therapeutic siRNAs. Trends Pharmacol. Sci. 25, 22–28.

    Article  PubMed  CAS  Google Scholar 

  4. Kamath, R. S., Fraser, A. G., Dong, Y., et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237.

    Article  PubMed  CAS  Google Scholar 

  5. Kamath, R. S. and Ahringer, J. (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321.

    Article  PubMed  CAS  Google Scholar 

  6. T. S., Myers, J. W., and Blau, H. M. (2004) Restriction enzyme-generated siRNA (REGS) vectors and libraries. Nat. Genet. 36, 183–189.

    Article  Google Scholar 

  7. Luo, B., Heard, A. D., and Lodish, H. F. (2004) Small interfering RNA production by enzymatic engineering of DNA (SPEED). Proc. Natl. Acad. Sci. USA 101, 5313–5314.

    Article  Google Scholar 

  8. Berns, K., Hijmans, E. M., Mullenders, J., et al. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437.

    Article  PubMed  CAS  Google Scholar 

  9. De Backer, M. D., Nelissen, B., Logghe, M., et al. (2001) An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans. Nat. Biotechnol. 19, 235–241.

    Article  PubMed  Google Scholar 

  10. Kawasaki, H., Onuki, R., Suyama, E., and Taira, K. (2002) Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries. Nat. Biotechnol. 20, 376–380.

    Article  PubMed  CAS  Google Scholar 

  11. Zheng, L., Batalov, S., Zhou, D., Orth, A., Ding, S., and Schultz, P. G. (2004) An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 135–140.

    Article  PubMed  CAS  Google Scholar 

  12. Sioud, M. (2004) Therapeutic potential of small interfering RNAs. Drugs of the Future, 29, 741–750.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Norwegian Cancer Society. We thank Dr. Anne Dybwad for critical reading of the manuscript.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Sioud, M., Wälchli, S. (2005). Strategies for the Design of Random siRNA Libraries and the Selection of anti-GFP siRNAs. In: Carmichael, G.G. (eds) RNA Silencing. Methods in Molecular Biology™, vol 309. Humana Press. https://doi.org/10.1385/1-59259-935-4:083

Download citation

  • DOI: https://doi.org/10.1385/1-59259-935-4:083

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-436-4

  • Online ISBN: 978-1-59259-935-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics