Production of highly potent recombinant siRNAs in Escherichia coli

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line
  • Chromatography, High Pressure Liquid
  • Escherichia coli / genetics*
  • Gene Expression
  • Gene Knockdown Techniques*
  • HeLa Cells
  • Humans
  • Plasmids
  • RNA Interference*
  • RNA, Bacterial / chemistry
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics*


  • RNA, Bacterial
  • RNA, Small Interfering