CCR RNAi initiative: Technology and assay development

对全球监测系统和CCR RNAi倡议的工作至关重要的是利用和开发新技术和分析方法。RNA分析：我们现在已经为我们的商业RNA分析(“Quantigene”，Genopsectra Inc.)进行了充分的评估和优化。这使我们能够在相对高通量的RNA水平上进行RNAi分析。我们现在已经利用“量子基因”分析方法分析了100多个人类基因的RNAi。蛋白质分析：在2005年间，我们与Paul Goldsmith博士和John Weinstein博士合作开发了一种在蛋白质水平上进行RNAi分析的定量方法。这种基于蛋白质印迹的检测系统给出了重复性很高的结果。细胞系和转染优化：开发筛选系统以验证RNAi资源的关键是确定最适合进行这项工作的细胞系(S)。我们通常在人结肠癌细胞系HCT-116或人乳腺癌细胞系MDA-MB-231中进行大多数RNAi分析。不过，我们也调整了我们的转基因方案，使其适用于另外10多个细胞系。此外，这一过程的很大一部分已经自动化，以减少错误并显著提高重复性。功能分析：我们现在正在增加我们的承诺，使最合适的功能分析与RNAi分析保持一致，这将成为我们2006年工作的一个越来越重要的部分。

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.