siRNA Design pp 193-204 | Cite as

Designing Efficient and Specific Endoribonuclease-Prepared siRNAs

  • Vineeth Surendranath
  • Mirko Theis
  • Bianca H. Habermann
  • Frank BuchholzEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 942)


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.

Key words

RNA interference esiRNA siRNA Off-target effects siRNA pool siRNA efficiency 


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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Vineeth Surendranath
    • 1
  • Mirko Theis
    • 1
  • Bianca H. Habermann
    • 2
  • Frank Buchholz
    • 3
    Email author
  1. 1.Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany
  2. 2.Max Planck Institute for Biology of AgeingCologneGermany
  3. 3.Department of Medical Systems BiologyUniversity Cancer Center, University Hospital and Medical Faculty Carl Gustav Carus, University of Technology DresdenDresdenGermany

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