chapter  6
Production of siRNA In Vitro by Enzymatic Digestion of Double-Stranded RNA
WithFrank Buchholz, David Drechsel, Martine Ruer, Ralf Kittler
Pages 14

RNAi is an ancient defense and regulatory mechanism to silence genes in a variety of organisms, including plants, insects and vertebrates (reviewed in references1,2). The discovery of this mechanism has enabled researchers to utilize it as a tool for gene function analyses in model organisms. Its relative ease has generated a wealth of new information, and for some organisms whole genome RNAi analyses have been accomplished.3-5 In many species, long dsRNA can be used to trigger RNAi when introduced into the cytoplasm of a cell. However, long dsRNAs have a nonspecific negative effect on cell proliferation in many vertebrate cells because they trigger an interferon response (reviewed inreference [6]). Interferon production activates two pathways in the cell that negatively affect cell proliferation. First, interferons cause the activation of dsRNAdependent protein kinase (PKR), leading to the general inhibition of protein production by phosphorylation of the translation factor eIF-2α. Second, 2'-5' oligoadenylate synthetase (2'-5'-OAS) is turned on, leading to mRNA degradation via activation of RNase L. As a consequence, long dsRNA is not useful for specific gene silencing in most mammalian cells.