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Production of highly potent recombinant siRNAs in Escherichia coli.

Publication ,  Journal Article
Huang, L; Lieberman, J
Published in: Nature protocols
December 2013

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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Published In

Nature protocols

DOI

EISSN

1750-2799

ISSN

1754-2189

Publication Date

December 2013

Volume

8

Issue

12

Start / End Page

2325 / 2336

Related Subject Headings

  • RNA, Small Interfering
  • RNA, Bacterial
  • RNA Interference
  • Plasmids
  • Humans
  • Hela Cells
  • HeLa Cells
  • Gene Knockdown Techniques
  • Gene Expression
  • Escherichia coli
 

Citation

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Huang, L., & Lieberman, J. (2013). Production of highly potent recombinant siRNAs in Escherichia coli. Nature Protocols, 8(12), 2325–2336. https://doi.org/10.1038/nprot.2013.149
Huang, Linfeng, and Judy Lieberman. “Production of highly potent recombinant siRNAs in Escherichia coli.Nature Protocols 8, no. 12 (December 2013): 2325–36. https://doi.org/10.1038/nprot.2013.149.
Huang L, Lieberman J. Production of highly potent recombinant siRNAs in Escherichia coli. Nature protocols. 2013 Dec;8(12):2325–36.
Huang, Linfeng, and Judy Lieberman. “Production of highly potent recombinant siRNAs in Escherichia coli.Nature Protocols, vol. 8, no. 12, Dec. 2013, pp. 2325–36. Epmc, doi:10.1038/nprot.2013.149.
Huang L, Lieberman J. Production of highly potent recombinant siRNAs in Escherichia coli. Nature protocols. 2013 Dec;8(12):2325–2336.

Published In

Nature protocols

DOI

EISSN

1750-2799

ISSN

1754-2189

Publication Date

December 2013

Volume

8

Issue

12

Start / End Page

2325 / 2336

Related Subject Headings

  • RNA, Small Interfering
  • RNA, Bacterial
  • RNA Interference
  • Plasmids
  • Humans
  • Hela Cells
  • HeLa Cells
  • Gene Knockdown Techniques
  • Gene Expression
  • Escherichia coli