Protocol

RNA Silencing

Volume 309 of the series Methods in Molecular Biology™ pp 93-196

Silencing Gene Expression with Dicer-Generated siRNA Pools

  • Jason W. MyersAffiliated withDepartment of Molecular Pharmacology, Stanford University Medical School
  • , James E. FerrellJr.Affiliated withDepartments of Molecular Pharmacology and Biochemistry, Stanford University Medical School

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Abstract

With the explosion of genomic information, there is an increasing need to analyze gene function in a high-throughput fashion. This makes reverse genetic approaches extremely attractive; however, in most mammalian and vertebrate systems it has been difficult and time-consuming to develop a cellular model deficient in one or more proteins. The discovery of RNA interference (RNAi) has made loss-of-function studies relatively quick and easy and amenable to high-throughput formats (13). Double-stranded RNAs (dsRNAs) 21 nucleotides in length, known as small interfering RNAs (siRNAs), are introduced into the cytosol, where they are unwound (4), allowing the antisense strand to interact in a sequence-specific manner with the complementary mRNA (5). Binding of the antisense strand to the target mRNA triggers cleavage of the mRNA (6). siRNA-mediated gene silencing is very specific, most likely because of the high specificity of nucleotide base-pairing.