High-throughput selection of effective RNAi probes for gene silencing
High-throughput selection of effective RNAi probes for gene silencing
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DOI：
10.1101/gr.1575003
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发表时间：
2003-10-01
期刊：
影响因子：
7
通讯作者：
Mittal, V
中科院分区：
文献类型：
--
作者：
Kumar, R;Conklin, DS;Mittal, V
RNA干扰（RNA interference，RNAi）是由双链RNA介导的序列特异性转录后基因沉默过程。RNA！最近成为一个强大的遗传工具，分析基因功能的哺乳动物细胞。然而，这种方法的能力是有限的，在预测小干扰RNA（siRNA）沉默基因的功效的不确定性。这不仅对小规模而且对哺乳动物系统中的高通量RNAi筛选倡议施加了严重的限制。我们已经开发了一种可靠的定量方法，用于快速有效地鉴定针对任何基因的最有效的siRNA。siRNA序列的功效通过其降低同源靶-报告基因融合体的表达的能力来监测，其具有容易定量的读数。最后，使用基于微阵列的细胞转染，我们证明了这种方法在高通量筛选中的无限潜力，用于识别有效的siRNA探针，用于沉默哺乳动物系统中的基因。这种方法可能会影响RNAi作为一种反向遗传工具，用于在全基因组范围内分析哺乳动物基因功能。
RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNA! has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using microarray-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.