Harnessing RNAi for Analyses of Ras Signaling and Transformation

The Ras-regulatory network is a loosely defined composition of numerous Ras family members and effector pathways that couple to critical cell-regulatory processes. Investigators are increasingly turning to RNAi-mediated inhibition of gene expression as an effective tool to help generate authentic portraits of Ras protein function in general and to accurately characterize the contribution of Ras family members and Ras effectors to oncogenic transformation in particular. Here we provide detailed protocols for high-efficiency and high-throughput delivery of siRNAs to human cancer cell lines and primary human epithelial cells. In addition, we discuss appropriate controls and limitations for the use of RNAi to derive biologically relevant observations.