Production of Endoribonuclease-Prepared Short Interfering RNAs (esiRNAs) for Specific and Effective Gene Silencing in Mammalian Cells.
Production of Endoribonuclease-Prepared Short Interfering RNAs (esiRNAs) for Specific and Effective Gene Silencing in Mammalian Cells.
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DOI：
10.1101/pdb.prot4824
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发表时间：
2007-08-01
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影响因子：
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通讯作者：
Buchholz, Frank
中科院分区：
文献类型：
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作者：
Heninger, Anne-Kristin;Buchholz, Frank
RNA干扰机制已成为研究许多生物体功能丧失的实用工具。为了使基因敲除方法适用于哺乳动物细胞，需要应用短干扰双链RNA(SiRNAs)。该方案描述了一种简单、低成本的方法，该方法使用重组大肠杆菌RNaseIII将长dsRNA消化成内切酶准备的短干扰RNA(EsiRNAs)。这项技术的优点是所产生的esiRNA的高效率和特异性，它不仅适用于小规模的应用，而且还适用于高通量的功能损失分析。EsiRNA的另一个伟大资产是它在设计上的灵活性，使用基于Web的工具，如DEQOR，或来自数据库谜语的预先设计的esiRNA序列。产生带有T7启动子序列的聚合酶链式反应产物，对其进行转录和退火化。产生的长dsRNA被酶消化成重叠的esiRNA池，随后被旋转柱纯化。
INTRODUCTIONThe mechanism of RNA interference has emerged as a practical tool to study loss of function in many organisms. To make the method of gene knockdown suitable for mammalian cells, short interfering double-stranded RNAs (siRNAs) need to be applied. This protocol describes a straightforward, low-cost method, which uses recombinant Escherichia coli RNase III to digest long dsRNA into endoribonuclease-prepared short interfering RNAs (esiRNAs). Advantages of this technology are the high efficiency and specificity of the resulting esiRNA and its usefulness for not only small-scale applications, but also high-throughput loss-of-function analyses. Another great asset of esiRNA is its flexibility in design, using Web-based tools such as DEQOR, or predesigned esiRNA sequences from the database RiDDLE. PCR products flanked with T7 promoter sequences are generated, transcribed, and annealed. The resulting long dsRNA is enzymatically digested into a pool of overlapping esiRNAs, which are subsequently spin-column-purified.