Large-scale loss of function RNAi screening has the greatest potential for the discovery of novel gene function and the identification of new protein targets with clinical relevance. Building upon our experience using synthetic siRNAs to down-regulate individual genes (ZO1 BC 010613) we have established robust RNAi screening formats using synthetic siRNAs. Developing an RNAi screening capability has required extensive optimization of the induction of RNAi by synthetic siRNAs in a manner compatible with a larger scale workflow that generates robust and reproducible data. Critical to establishing effective RNAi screening has been the development of optimized protocols, statistical analysis, and down-stream validation procedures. The scale of the RNAi screens we have conducted has started at a modest level, targeting 400 to 500 genes but in FY09 this has expanded to enable us to simultaneously analyze several thousand genes. Our independent and collaborative studies have established conditions for synthetic siRNA-mediated RNAi screening in several cancer cell lines, including those used for the study of breast, ovarian, and colorectal cancer. Independent and collaborative RNAi screens to identify novel cancer-associated genes, including genes that can be exploited directly as anti-cancer molecular targets have been initiated. This hypothesis-generating approach has enabled us to identify a number of proteins that influence the growth of cancer cell lines and follow-up analysis of these specific proteins is on-going.Large-scale loss of function RNAi screening has the greatest potential for the discovery of novel gene function and the identification of new protein targets with clinical relevance. Building upon our experience using synthetic siRNAs to down-regulate individual genes (ZO1 BC 010613) we have established robust RNAi screening formats using synthetic siRNAs. Developing an RNAi screening capability has required extensive optimization of the induction of RNAi by synthetic siRNAs in a manner compatible with a larger scale workflow that generates robust and reproducible data. Critical to establishing effective RNAi screening has been the development of optimized protocols, statistical analysis, and down-stream validation procedures. The scale of the RNAi screens we have conducted has started at a modest level, targeting 400 to 500 genes but in FY09 this has expanded to enable us to simultaneously analyze several thousand genes. Our independent and collaborative studies have established conditions for synthetic siRNA-mediated RNAi screening in several cancer cell lines, including those used for the study of breast, ovarian, and colorectal cancer. Independent and collaborative RNAi screens to identify novel cancer-associated genes, including genes that can be exploited directly as anti-cancer molecular targets have been initiated. This hypothesis-generating approach has enabled us to identify a number of proteins that influence the growth of cancer cell lines and follow-up analysis of these specific proteins is on-going.
