Surendranath Vineeth, Theis Mirko, Habermann Bianca H, Buchholz Frank
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
Methods Mol Biol. 2013;942:193-204. doi: 10.1007/978-1-62703-119-6_11.
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.
RNA干扰（RNAi）已发展成为功能丧失研究的主要技术之一，有助于阐明各种细胞系统和模式生物中基因的生物学功能。对于许多无脊椎动物，如黑腹果蝇（D. melanogaster）和秀丽隐杆线虫（C. elegans），长双链RNA（dsRNA）可直接用于诱导RNAi反应，而在哺乳动物细胞中通常使用化学合成的小干扰RNA（siRNA），以避免长dsRNA引发的类似干扰素的反应（Reynolds等人，《RNA》12：988 - 993，2006）。然而，siRNA价格昂贵，且存在非故意基因靶向效应（脱靶效应），这会混淆此类研究结果的分析。我们和其他研究人员已经开发出一种用于哺乳动物细胞RNAi的替代技术，称为核糖核酸酶制备的siRNA（esiRNA），它基于在体外通过重组核糖核酸酶III消化长dsRNA来酶促生成siRNA池（Yang等人，《美国国家科学院院刊》99：9942 - 9947，2002；Myers等人，《自然生物技术》21：324 - 328，2003）。该技术已被证明具有成本效益且可靠。此外，多项研究表明，esiRNA中固有的靶向一个转录本的复杂siRNA池可减少脱靶效应（Myers等人，《RNA干扰与基因沉默》2：181，2006；Kittler等人，《自然方法》4：337 - 344，2007）。在本章中，我们描述了生成靶向优化esiRNA的设计标准。
