Du Quan, Thonberg Håkan, Zhang Hong-Yan, Wahlestedt Claes, Liang Zicai
Center for Genomics and Bioinformatics, Karolinska Institutet, 171 77 Stockholm, Sweden.
Biochem Biophys Res Commun. 2004 Dec 3;325(1):243-9. doi: 10.1016/j.bbrc.2004.09.222.
Only a small fraction of all siRNAs are effective in silencing their target genes, and siRNA efficacy can only be determined experimentally. Previously described reporter-based siRNA validation methods all rely on the availability of physical cDNA clones, and this limits the high throughput applicability of the method. In the current report, we used short synthetic DNA fragment containing a siRNA targeting site, instead of cDNA, to fuse with a reporter gene. When targeting such transcripts with different siRNAs, we found that such constructs can faithfully report the efficacy of the corresponding siRNAs in a sequence specific manner, even when the inserted DNA fragment is essentially only long enough to cover the targeting site. The efficacy of both vector-based siRNA and synthetic siRNA can be evaluated using this system. Since only readily available short synthetic DNA fragments are needed for forming the evaluation vector, this method provides an appealing way of validating siRNAs in high throughput.
所有小干扰RNA（siRNA）中只有一小部分能够有效地沉默其靶基因，且siRNA的有效性只能通过实验来确定。先前描述的基于报告基因的siRNA验证方法均依赖于物理cDNA克隆的可用性，这限制了该方法的高通量适用性。在本报告中，我们使用了含有siRNA靶向位点的短合成DNA片段，而非cDNA，来与报告基因融合。当用不同的siRNA靶向此类转录本时，我们发现这种构建体能够以序列特异性的方式如实地报告相应siRNA的有效性，即使插入的DNA片段基本上仅足够长以覆盖靶向位点。基于载体的siRNA和合成siRNA的有效性均可使用该系统进行评估。由于形成评估载体仅需要容易获得的短合成DNA片段，因此该方法为高通量验证siRNA提供了一种有吸引力的方式。
