CCR RNAi initiative: Technology and assay development

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Critical to the work of GSS and the CCR RNAi initiative has been the utilization and development of new technologies and assays. RNA analysis: We have now fully assessed and optimized for our use a commercial RNA assay ("Quantigene", Genopsectra Inc.) that allows us to conduct RNAi analysis at an RNA level in relatively high-throughput. We have now utilized the "Quantigene" assay to analyze RNAi for over 100 human genes. Protein analysis: During 2005 we have developed in collaboration with Dr. Paul Goldsmith and Dr. John Weinstein a quantitative approach to RNAI analysis at a protein level. This Western blot based assay system is giving highly reproducible results. Cell lines and transfection optimization: Critical to developing a screening system to validating RNAi resources has been the determination of the most suitable cell line(s) in which to conduct this work. We routinely conducted most RNAi analysis in either a human colon cancer cell line HCT-116 or a human breast cancer cell line MDA-MB-231. We have, though also adapted our transfection protocol to over 10 additional cell lines. In addition, a significant part of this procedure has automated to reduce error and significantly enhance reproducibility. Functional analysis: We are now increasing our commitment to aligning the most suitable functional assays with RNAi analysis and this will become an increasingly important part of our work over 2006.