Production of highly potent recombinant siRNAs in Escherichia coli.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Huang, L; Lieberman, J

Published Date

  • December 2013

Published In

Volume / Issue

  • 8 / 12

Start / End Page

  • 2325 - 2336

PubMed ID

  • 24177290

Electronic International Standard Serial Number (EISSN)

  • 1750-2799

International Standard Serial Number (ISSN)

  • 1754-2189

Digital Object Identifier (DOI)

  • 10.1038/nprot.2013.149

Language

  • eng