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          Institute: MPI für molekulare Zellbiologie und Genetik     Collection: MPI-CBG Publications 2013 (arch)     Display Documents



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ID: 688447.0, MPI für molekulare Zellbiologie und Genetik / MPI-CBG Publications 2013 (arch)
Designing efficient and specific endoribonuclease-prepared siRNAs.
Authors:Surendranath, Vineeth; Theis, Mirko; Habermann, Bianca; Buchholz, Frank
Date of Publication (YYYY-MM-DD):2013
Title of Journal:Methods in Molecular Biology (Clifton, N.J.)
Volume:942
Start Page:193
End Page:204
Copyright:not available
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:RNA interference (RNAi) has grown to be one of the main techniques for loss-of-function studies, leading to the elucidation of biological function of genes in various cellular systems and model organisms. While for many invertebrates such as Drosophila melanogaster (D. melanogaster) and Caenorhabditis elegans (C. elegans) long double-stranded RNA (dsRNA) can directly be used to induce a RNAi response, chemically synthesized small interfering RNAs (siRNAs) are typically employed in mammalian cells to avoid an interferon-like response triggered by long dsRNA (Reynolds et al., RNA 12:988-993, 2006). However, siRNAs are expensive and beset with unintentional gene targeting effects (off-targets) confounding the analysis of results from such studies. We, and others, have developed an alternative technology for RNAi in mammalian cells, termed endoribonuclease-prepared siRNA (esiRNA), which is based on the enzymatic generation of siRNA pools by digestion of long dsRNAs with recombinant RNase III in vitro (Yang et al., Proc Natl Acad Sci USA 99: 9942-9947, 2002; Myers et al., Nat Biotechnol 21:324-328; 2003). This technology has proven to be cost-efficient and reliable. Furthermore, several studies have demonstrated that complex pools of siRNAs, as inherent in esiRNAs, which target one transcript reduce off-target effects (Myers et al., J RNAi Gene Silencing 2:181, 2006; Kittler et al., Nat Methods 4:337-344, 2007). Within this chapter we describe design criteria for the generation of target-optimized esiRNAs.
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:Lib
Affiliations:MPI für molekulare Zellbiologie und Genetik
Identifiers:LOCALID:5093
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