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. This has proven to be highly compatible with focused RNAi screening studies investigating a specific pathway, process or protein family. 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. Plans are now in development to enhance the throughput and scale of our siRNA screening capacity to target several thousands of human genes.
| Martin, Scott E; Jones, Tamara L; Thomas, Cheryl L et al. (2007) Multiplexing siRNAs to compress RNAi-based screen size in human cells. Nucleic Acids Res 35:e57 |
| Martin, Scott E; Caplen, Natasha J (2007) Applications of RNA interference in mammalian systems. Annu Rev Genomics Hum Genet 8:81-108 |
| Martin, Scott E; Caplen, Natasha J (2006) Mismatched siRNAs downregulate mRNAs as a function of target site location. FEBS Lett 580:3694-8 |
| Ludwig, Joseph A; Szakacs, Gergely; Martin, Scott E et al. (2006) Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer. Cancer Res 66:4808-15 |
| Dombroski, Derek; Houghtling, Richard A; Labno, Christine M et al. (2005) Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton. J Immunol 174:1385-92 |
| Huppi, Konrad; Martin, Scott E; Caplen, Natasha J (2005) Defining and assaying RNAi in mammalian cells. Mol Cell 17:1-10 |
