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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)

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

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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
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  2. Anne-Kristin Heninger
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  3. Kristin Franke
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  4. Bianca Habermann
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  5. Frank Buchholz
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Correspondence to Frank Buchholz.

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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

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