Production of highly potent recombinant siRNAs in Escherichia coli.
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.
Duke Scholars
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Related Subject Headings
- RNA, Small Interfering
- RNA, Bacterial
- RNA Interference
- Plasmids
- Humans
- Hela Cells
- HeLa Cells
- Gene Knockdown Techniques
- Gene Expression
- Escherichia coli
Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- RNA, Small Interfering
- RNA, Bacterial
- RNA Interference
- Plasmids
- Humans
- Hela Cells
- HeLa Cells
- Gene Knockdown Techniques
- Gene Expression
- Escherichia coli