Microarrays for the large-scale functional analysis of genes by RNA interference

本研究的目的是开发允许平行转染多个sirna的微阵列，用于基于细胞的基因功能高通量分析。因为转染效率是决定这种微阵列有用性的最关键因素之一，我们主要参与了提高转染效率的siRNA阵列的制备。首先，我们尝试制备siRNA微阵列，其方法是利用在金蒸发玻璃板上形成的微图案自组装单层，将siRNA和阳离子脂质（转染增强子）组成的复合物以阵列形式加载。通过将HEK293细胞直接植入同时装载egfp编码质粒和egfp靶向siRNA的微阵列，验证了该方法的可行性。这项研究的结果已提交给国际期刊发表。此外，该方法也适用于将cDNA体外转录成长双链RNA，然后用核酸内切酶切酶（dicer）酶切而成的siRNA的转染。由于dicer-processed RNA含有多种寡核苷酸，因此不需要选择siRNA靶向的最有效序列之一。这个特性使得siRNA阵列更有用。为了提高转染效率，我们研究了另一种系统，其中电脉冲应用于将负载的siRNA从阵列中分离并引入细胞（电穿孔）。通过优化阵列制备条件和电脉冲，可以实现siRNA向直接培养细胞的高效转移。

The aim of this study was to develop microarrays that allow the parallel transfection of multiple siRNAs for the cell-based high-throughput analysis of gene functions. Because transfection efficiency is one of the most crucial factors that determine the usefulness of such microarrays, we were mostly involved in the preparation of siRNA arrays with improved transfection efficiency.First we made an attempt to prepare siRNA microarrays in the way that complexes consisting of siRNA and a cationic lipid (transfection enhancer) were loaded in an array format taking advantage of micropatterned, self-assembled monolayers formed on a gold-evaporated glass plate. The feasibility of the method was demonstrated by directly seeding HEK293 cells to the microarray on which EGFP-coding plasmid and EGFP-targeting siRNA were simultaneously loaded. The result of this study was submitted for publication in the international journal.In addition, the method could also be applied to the transfection of siRNA that was prepared by in vitro transcription of cDNA into long double-stranded RNA followed by digestion with endonuclease, dicer. Because dicer-processed RNA contains a variety of oligonucleotides, it is not necessary to select one of the most effective sequences targeted by siRNA. This feature makes the siRNA array more useful.In order to enhance much more transfection efficiency, we studied another system in which an electric pulse was applied to detach the loaded siRNA from the array to introduce into cells (electroporation). By optimizing conditions for array preparation and electric pulsing, the highly-efficient transfer of siRNA to the directly-cultured cells could be achieved.