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Production of highly potent recombinant siRNAs in Escherichia coli

Nature Protocols volume 8, pages 2325–2336 (2013)Cite this article

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Abstract

We recently invented a method to produce highly potent siRNAs in Escherichia coli, based on the serendipitous discovery that ectopic expression of p19, a plant viral siRNA-binding protein, stabilizes otherwise unstable bacterial siRNAs, which we named pro-siRNAs for prokaryotic siRNAs. We present a detailed protocol describing how to produce pro-siRNAs for efficiently knocking down any gene, beginning with the design of a pro-siRNA expression plasmid and ending with siRNA purification. This protocol uses one plasmid to co-express a recombinant His-tagged p19 protein and a long hairpin RNA containing sense and antisense sequences of the target gene. pro-siRNAs are isolated and purified using nickel beads and HPLC, using methods used to produce recombinant proteins. Once a pro-siRNA plasmid is obtained, production of purified pro-siRNAs takes a few days. The pro-siRNA technique provides a reliable and renewable source of siRNAs, and it can be implemented in any laboratory whose members are skilled in routine molecular biology techniques.

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Figure 1: Workflow of the protocol to produce pro-siRNAs from E. coli.
Figure 2: Design and analysis of pro-siRNA plasmid.
Figure 3: Purification of pro-siRNA.

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Acknowledgements

We thank J. Carrington (Donald Danforth Plant Science Center) for providing the p19 clone. We also thank J. Jin and L. McReynolds from New England Biolabs and P. Deighan and E. Kiner from Harvard Medical School for assistance and advice in establishing this method. We also thank Lieberman Laboratory members for technical assistance. This work was supported by a US National Institutes of Health grant (no. AI087431; J.L.) and a GlaxoSmithKline(GSK)-Immune Disease Institute(IDI) Alliance fellowship (L.H.).

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Authors and Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA

    Linfeng Huang & Judy Lieberman

  2. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

    Linfeng Huang & Judy Lieberman

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  1. Linfeng Huang
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  2. Judy Lieberman
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Contributions

L.H. designed the protocol with suggestions from J.L. and L.H., and J.L. wrote the paper.

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Correspondence to Linfeng Huang or Judy Lieberman.

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Huang, L., Lieberman, J. Production of highly potent recombinant siRNAs in Escherichia coli. Nat Protoc 8, 2325–2336 (2013). https://doi.org/10.1038/nprot.2013.149

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