CCR RNAi Initiative: Technology and Assay Development

利用内源性RNA为基础的基因沉默机制，RNA干扰（RNAi）技术迅速发展，用于解剖基因-功能关系，并作为进一步分子靶标分析的手段。然而，由特定RNAi效应物介导的基因特异性效应很难预测，并且在文献中经常报道不足。我们着手建立强大的分析和基于细胞系的模型系统，以便快速和可重复地评估RNAi对基因特异性转录物和蛋白质水平的影响。我们最初专注于评估由合成sirna群体介导的基因沉默。我们现在已经充分评估和优化了我们使用的商业RNA分析（Quantigene, Genopsectra Inc.），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Quantigene分析超过100个人类基因的RNAi，并且还使用基于Western blot的分析系统和ECL蛋白质分析方法来量化19种人类蛋白质在蛋白质水平上介导的敲低。这项分析的结果构成了最近发表在《核酸研究》上的一篇文章的关键部分。该数据集是公共领域中最大的同类数据集之一。我们还扩展了我们的RNAi分析，以利用Quantigene检测系统的多重版本；初步结果非常令人鼓舞。大量的研究已经证明了在人类细胞中使用大通量、高通量RNAi筛选的价值。开发有效的RNAi筛选的关键是开发合适的高通量格式、分析、统计分析和下游验证程序。我们已经进行了几项独立和合作研究，以建立几种不同癌细胞系的RNAi筛选条件，包括那些常规用于乳腺癌，卵巢癌和结直肠癌研究的细胞系。一个RNAi筛选使用多重方法，将sirna与多个基因结合，以压缩筛选的大小。在乳腺癌细胞系中进行的这种筛选确定了许多已知和假定的抗癌分子靶点；这项研究的结果发表在《核酸研究》杂志上。在开发了优化条件后，我们研究了各种独立和协作的RNAi筛选，以鉴定新的癌症相关基因，包括可以直接用作抗癌分子靶点的基因。这种假设生成方法已经确定了一些影响癌细胞系生长的蛋白质，这是我们独立和合作的RNAi筛选的结果，并正在进行后续分析，以调查这些特定蛋白质。

Technologies that exploit the endogenous RNA-based gene silencing mechanism, RNA interference (RNAi), have developed rapidly for the dissection of gene-function relationships and as a means of furthering molecular target analysis. However, the gene-specific effects mediated by a particular RNAi effector is difficult to predict and is frequently poorly reported in the literature. We set out to establish robust assays and cell line based model systems to enable rapid and reproducible assessment of the effects of RNAi on gene-specific transcript and protein levels. We initially focused on the assessment of the gene silencing mediated by a population of synthetic siRNAs. 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 and have also used a Western blot based assay system and an ECL protein analysis method to quantify the knockdown mediated at a protein level for 19 human proteins. The results of this analysis form a key part of a recently published article in Nucleic Acids Research. This data set represents one of the largest of its kind available in the public domain. We have also extended our RNAi analysis to make use of a multiplex version of the Quantigene assay system; initial results are very encouraging. The value of large, high-throughput RNAi screens in human cells has been demonstrated in a number of studies. Critical to developing effective RNAi screening has been the development of suitable high-throughput formats, assays, statistical analysis and down-stream validation procedures. We have conducted several Independent and collaborative studies to establish conditions for RNAi screening in several different cancer cell lines including those used routinely for studies of breast, ovarian, and colorectal cancer. One RNAi screen conducted using a multiplex approach that combined siRNAs to multiple genes so as to compress the size of the screen. This screen, conducted in a breast cancer cell line identified a number of known and putative anti-cancer molecular targets; the results of this study have been published in Nucleic Acids Research. Having developed optimized conditions we have studied a variety of independent and collaborative RNAi screens to identify novel cancer associated genes and including genes that can be exploited directly as anti-cancer molecular targets have now been performed. This hypothesis generating approach has identified a number of proteins that influence the growth of cancer cell lines as a result of our independent and collaborative RNAi screens and follow up analysis to investigate these specific proteins are on going.