RNAi-mediated knockdown of protein expression

Abstract

RNA interference is an essential method for studying genomic functions of single genes by loss-of-function experiments. Short interfering siRNAs are efficiently transfected into cultured cells to enable RISC-mediated mRNA cleavage and inhibition of translation in a sequence-specific manner. RNAi enables knockdown of single genes and screening for specific cellular processes or outcomes. In this chapter, we describe a detailed universal protocol for lipoplex-mediated siRNA transfection for cell cultures and cell lysis for subsequent RNA or protein analysis. The experimental procedure is described for verification of knockdown and includes cell lysis for mRNA or protein quantification. Important aspects for specific gene silencing and potential pitfalls are discussed.