Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
Methods. 2011 Apr;53(4):424-9. doi: 10.1016/j.ymeth.2010.12.021. Epub 2010 Dec 23.
The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.
先进的功能基因组工具的发展为系统研究健康和疾病中的生物学过程铺平了道路。特别是，RNA 干扰 (RNAi) 的实施作为一种全基因组、功能丧失筛选工具，使科学家能够在细胞测定中探测每个基因的作用，并且近年来，利用 RNAi 筛选已经发现了许多新的各种过程的因素。然而，这些结果也表明了生物系统的复杂性，并表明我们离深入理解功能网络还有很长的路要走。尽管如此，RNAi 筛选仍是一种强大的高通量方法，可以研究基因功能，并且已经开发出了多种在哺乳动物细胞中引发 RNAi 的方法。在这里，我们描述了在使用内切核酸酶制备的 (e)siRNAs 进行 RNAi 筛选时应考虑的步骤。我们提供了有关如何实施筛选和分析结果的有用信息。此外，我们讨论了命中验证的策略，并提出了一个关于如何跟踪验证命中以获得对潜在表型的分子理解的概述。
