High-throughput RNA interference
Introduction RNA interference (RNAi) is an evolutionarily conserved pathway of gene silencing that identifies and destroys mRNA sequences derived from selfish repetitive or viral sequences. The mechanisms and physiologic functions of RNAi pathways are discussed in Section 1. Because RNAi can be triggered by exogenously supplied double-stranded RNA (dsRNA) molecules, in principle one can effectively silence the expression of a gene and infer its physiologic function given its sequence. RNAi analysis on a genome-wide scale is a versatile and powerful tool that holds great promise in functional annotation of the human genome and in acceleration of drug target discovery and validation. The rapid advances to date have illustrated that high-throughput RNAi can complement genetic studies in traditional model organisms to extend our understanding and probe the mechanisms of well-studied pathways. In this chapter, we will focus on emerging methodologies and issues surrounding high-throughput RNAi in mammalian systems, and highlight the potential advances and pitfalls with these new technologies. Generation of genome-wide RNAi reagents In order to silence each gene in the genome using RNAi, the first step is to create the appropriate library of dsRNA reagents. In Caenorhabditis elegans and Drosophila melanogaster, long segments of dsRNA are readily taken up by the animal or tissue culture cells, respectively, and genome-wide RNAi libraries can be constructed by annealing complementary RNAs synthesized from both strands of the same cDNA.
