Using RNAi screening technologies to interrogate the extrinsic apoptosis pathway

Despite the knowledge accumulated during the last two decades about programmed cell death, further investigations of the complex regulatory network of apoptosis, including the extrinsic pathways, are still needed to gain an exhaustive and comprehensive understanding of this critical biological process. In addition, the identification of novel modulators of apoptosis may represent a good opportunity for making new paths into an otherwise heavily investigated area, therefore providing a molecular basis for new therapeutic strategies. In the last decade, RNA interference has become the technology of choice for discovering genes that encode molecules with previously unknown functions in biological pathways of interest. Various RNAi reagents and library formats have been developed and harnessed for high-throughput screening technologies to enable almost limitless investigation to uncover gene functions and networks in the context of basic biology and biomedical research including cancer biology. Although RNAi screening has been demonstrated to be a very powerful tool, various caveats and pitfalls have been progressively uncovered, including, but not limited to the enduring off-target effects. As the novelty of its bells and whistles have begun to diminish, functional genomic screens have morphed into a specialized field within the high-throughput screening community, where expert investigators progressively establish rigorous strategies to mitigate most of its possible flaws. Using various examples of RNAi screens conducted to further understand the extrinsic apoptosis pathway, this chapter describes the different RNAi tools and screening formats available and reviews the parameters one has to critically consider in order to be successful in implementing this technology.