Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells

Nature Methods volume 2, pages 779–784 (2005)Cite this article

  • Agarose, electrophoresis grade (Invitrogen)

  • BIOTAQ Red DNA polymerase (Bioline)

  • Complementary DNA (cDNA) clones or cDNA preparations

  • Deoxynucleoside triphosphate (dNTP) mix (10 mM; Bioline)

  • dsRNA digestion buffer (20 mM Tris-HCl, 0.5 mM EDTA, 5 mM MgCl2, 1 mM Diothiothreitol (DTT), 140 mM NaCl, 2.7 mM KCl, 5% (vol/vol) glycerol (pH 7.9))

  • Elution buffer (20 mM Tris-HCl, 1 mM EDTA, 520 mM NaCl, (pH 8.0))

  • 70% (vol/vol) ethanol, cold

  • Equilibration buffer (20 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl (pH 8.0))

  • esiRNA loading dye (3 mg/ml Orange G, 5% (wt/vol) Ficoll; both from Sigma-Aldrich)

  • GST-RNase III (prepared as previously described)

  • Isopropanol

  • Marker DNA (for example, 25-bp DNA ladder, HyperLadder V; Bioline)

  • MEGAscript in vitro transcription kit (Ambion)

  • MgCl2 (50 mM; Bioline)

  • 10× ammonium reaction buffer (160 mM (NH4)2SO4, 670 mM Tris-HCl (pH 8.8), 0.1% (vol/vol) Tween 20; Bioline)

  • Primer oligonucleotides containing the T7 promoter sequence, as described in Step 2

  • Q Sepharose FastFlow (Amersham Biosciences)

  • Wash buffer, (20 mM Tris-HCl, 1 mM EDTA, 400 mM NaCl (pH 8.0))

Agarose, electrophoresis grade (Invitrogen)

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2: Selection of an optimal esiRNA target region with DEQOR.
Figure 3: Digestion of long dsRNA.

References

  1. Hannon, G.J. & Rossi, J.J. Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378 (2004).

    Article  CAS  Google Scholar 

  2. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  3. Paddison, P.J. et al. Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat. Methods 1, 163–167 (2004).

    Article  CAS  Google Scholar 

  4. Buchholz, F., Drechsel, D., Ruer, M. & Kittler, R. Production of siRNA in vitro by enzymatic digestion of double-stranded RNA. In Gene Silencing by RNA: Technology and Application (ed. Sohail, M.) 87–99 (CRC Press, Boca Raton, Florida, USA, 2004).

    Google Scholar 

  5. Kawasaki, H., Suyama, E., Iyo, M. & Taira, K. siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells. Nucleic Acids Res. 31, 981–987 (2003).

    Article  CAS  Google Scholar 

  6. Myers, J.W., Jones, J.T., Meyer, T. & Ferrell, J.E., Jr. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat. Biotechnol. 21, 324–328 (2003).

    Article  CAS  Google Scholar 

  7. Yang, D. et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 9942–9947 (2002).

    Article  CAS  Google Scholar 

  8. Yang, D., Goga, A. & Bishop, J.M. RNA interference (RNAi) with RNase III–prepared siRNAs. Methods Mol. Biol. 252, 471–482 (2004).

    CAS  PubMed  Google Scholar 

  9. Zhu, C. & Jiang, W. Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis. Proc. Natl. Acad. Sci. USA 102, 343–348 (2005).

    Article  CAS  Google Scholar 

  10. Kittler, R. et al. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036–1040 (2004).

    Article  CAS  Google Scholar 

  11. Calegari, F., Haubensak, W., Yang, D., Huttner, W.B. & Buchholz, F. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc. Natl. Acad. Sci. USA 99, 14236–14240 (2002).

    Article  CAS  Google Scholar 

  12. Henschel, A., Buchholz, F. & Habermann, B. DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Res. 32, W113–W120 (2004).

    Article  CAS  Google Scholar 

  13. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386 (2000).

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, D-01307, Germany

    Ralf Kittler, Anne-Kristin Heninger, Kristin Franke & Frank Buchholz

  2. Scionics Computer Innovation, GmbH, Pfotenhauerstrasse 110, Dresden, D-01307, Germany

    Bianca Habermann

Authors
  1. Ralf Kittler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anne-Kristin Heninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristin Franke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bianca Habermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frank Buchholz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frank Buchholz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kittler, R., Heninger, AK., Franke, K. et al. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nat Methods 2, 779–784 (2005). https://doi.org/10.1038/nmeth1005-779

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1005-779

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing
pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy