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Nat Protoc. 2013 Dec;8(12):2325-36. doi: 10.1038/nprot.2013.149. Epub 2013 Oct 31.

Production of highly potent recombinant siRNAs in Escherichia coli.

Author information

1
1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

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.

PMID:
24177290
DOI:
10.1038/nprot.2013.149
[Indexed for MEDLINE]

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