CCR RNAi Initiative: Technology and Assay Development

CCR RNAi 计划：技术和检测开发

• 批准号: 7592801
• 负责人: natasha caplen
• 金额: $ 34.91万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592801
• 关键词: Apoptosis; Biological Assay; Biological Models; Breast; Breast Cancer Cell; CCR; Cancer cell line; Cell Line; Cell Survival; Cells; Colorectal Cancer; Condition; Data Set; Development; Dissection; Gene Expression; Gene Silencing; Genes; Goals; Growth; Human; Literature; Malignant Neoplasms; Mediating; Methods; Molecular; Molecular Target; Nucleic Acids; Numbers; Ovarian; Population; Procedures; Process; Protein Analysis; Proteins; Protocols documentation; Public Domains; Publishing; RNA; RNA Interference; Reporting; Research; Screening procedure; Stream; System; Technology; Transcript; Validation; Western Blotting; anti-cancer therapeutic; assay development; base; clinically relevant; follow-up; gene function; improved; novel; numb protein; size

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.

利用基于内源RNA的基因沉默机制--RNA干扰(RNAi)的技术迅速发展，用于剖析基因与功能的关系，并作为进一步进行分子靶标分析的手段。然而，由特定的RNAi效应器介导的基因特异性效应很难预测，在文献中往往报道得很少。我们着手建立强大的分析和基于细胞系的模型系统，以实现对RNAi对基因特异性转录和蛋白质水平的影响的快速和可重复性的评估。我们最初专注于评估由一组合成的siRNAs介导的基因沉默。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，并使用了基于蛋白质印迹的分析系统和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除。这一分析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。这一数据集是公有领域可用的同类数据中最大的之一。我们还扩展了我们的RNAi分析，以利用量子基因分析系统的多路版本；初步结果非常令人鼓舞。大量研究已经证明了大型高通量RNAi筛选在人类细胞中的价值。开发有效的RNAi筛查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的研究，以建立在几种不同的癌细胞系中进行RNAi筛查的条件，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。使用将siRNA结合到多个基因以压缩屏幕大小的多重方法进行的一次RNAi筛选。这项在乳腺癌细胞系中进行的筛查确定了一些已知和假定的抗癌分子靶点；这项研究的结果已发表在《核酸研究》杂志上。在开发了优化的条件后，我们研究了各种独立和协作的RNAi筛选，以确定新的癌症相关基因，并包括可以直接用作抗癌分子靶点的基因。由于我们独立和合作的RNAi筛选，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，并正在进行后续分析，以研究这些特定的蛋白质。