High-throughput RNA interference strategies for target discovery and validation by using synthetic short interfering RNAs: functional genomics investigations of biological pathways.

摘要: During the past five years, RNA interference (RNAi) has emerged as arguably best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, readily upscalable methodologies. Originally applied experimentally in C. elegans Drosophila, RNAi is now widely used mammalian cell systems also. The development of commercially libraries short interfering RNAs (siRNAs) other silencing reagents targeting entire classes human provide opportunity carry out genome-scale screens discover characterize gene functions directly cells. A key challenge these studies, also faced by earlier or proteomics approaches, resides reaching an optimal balance necessarily high throughput desire achieve same level detailed analysis that routine conventional small-scale studies. This chapter discusses technical aspects how perform such screens, what parameters monitor, which readouts apply. Examples homogenous assays multiplexed high-content microscopy-based are demonstrated.