Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells

Ralf Kittler, Anne Kristin Heninger, Kristin Franke, Bianca Habermann, Frank Buchholz

Research output: Contribution to journalArticle

64 Scopus citations

Abstract

RNA interference (RNAi) has revolutionized functional genomic studies in many model organisms. In particular, the introduction of short double-stranded RNAs (dsRNAs) as mediators of RNAi in mammalian cells has moved loss-of-function studies in these experimental systems to a new plane1. Now the two most commonly used RNAi triggers in mammalian cells ar chemically or in vitro synthesized short interfeing RNAs (siRNAs)2 and in vivio expressed short hairpin RNAs (shRNAs)3. We, and others, have pioneered an alternative technology, which is considerably more cost-efficient and less laborious than siRNAs and shRNA methodologies. Our method is based on the generation of siRNAs by digestion of long dsRNAs with recombinant Escherichia coli RNase III or Dicer4-8. The resulting endoribonuclease-prepared siRNAs (esiRNAs) have proven to be efficient an specific mediators of RNAi in mammalian cells9-11. Here we describe a robust and simple protocol for the production of esiRNA (Fig. 1): a cDNA fragment tagged with T7 promoter sequence by PCR is transcribed in vitro to porduce dsRNA. By limited digestion, the long dsRNA is then converted to siRNAs of <30 base pairs (bp) and spin-purified in a single column. Because of its ease, speed and cost-efficiency, this protocol can be easily scaled for the generation of libraries for (sub)genomic screens.

Original languageEnglish (US)
Pages (from-to)779-784
Number of pages6
JournalNature methods
Volume2
Issue number10
DOIs
StatePublished - Oct 1 2005

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells'. Together they form a unique fingerprint.

Cite this