Modulation of Gene Expression by RNAi

RNA interference (RNAi) is a form of posttranscriptional gene silencing in which the presence within the cell of double-stranded RNA (dsRNA) leads to the specific degradation of mRNA with a complimentary sequence. RNAi is a natural phenomenon that can be exploited as a powerful tool to study gene function by generating gene “knockdowns” in various cell types. RNAi is mediated by short interfering RNAs (siRNAs), which are generated within cells from long dsRNAs. To avoid generalized toxic effects, mammalian cells are transfected directly with 21-23-bp-long siRNAs generated either by chemical synthesis or obtained by a series of enzymatic reactions. The present chapter deals with siRNA design, synthesis, trans-fection, and readout of efficiency in a mammalian cell culture system. The general principle is illustrated by the functional knockdown of p97/VCP (valosin-containing protein) in HeLa cells using five different siRNA sequences.