Kumar Rajeev, Conklin Douglas S, Mittal Vivek
Cancer Genome Research Center, Cold Spring Harbor Laboratory, Woodbury, New York 11797, USA.
Genome Res. 2003 Oct;13(10):2333-40. doi: 10.1101/gr.1575003.
RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi 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 micro array-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.
RNA干扰（RNAi）是一种由双链RNA介导的序列特异性转录后基因沉默过程。RNAi最近已成为分析哺乳动物细胞中基因功能的强大遗传工具。然而，这种方法的效力受到预测小干扰RNA（siRNA）沉默基因效力时不确定性的限制。这不仅给小规模的，也给哺乳动物系统中的高通量RNAi筛选计划带来了严重限制。我们开发了一种可靠且定量的方法，用于快速有效地鉴定针对任何基因的最有效siRNA。通过siRNA序列降低同源靶标 - 报告基因融合体表达的能力（具有易于量化的读数）来监测其效力。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具有无限潜力，可用于鉴定在哺乳动物系统中沉默基因的有效siRNA探针。这种方法可能对将RNAi用作在全基因组范围内分析哺乳动物基因功能的反向遗传工具具有重要意义。
