Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Biochem Biophys Res Commun. 2010 Mar 12;393(3):498-503. doi: 10.1016/j.bbrc.2010.02.033. Epub 2010 Feb 10.
The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.
小干扰 RNA（siRNA）的功效和特异性在很大程度上取决于 siRNA 序列。由于目前只能通过经验策略来预测这些参数，因此需要简单而准确的 siRNA 评估方法。为了简化这些实验，通常会使用带有报告基因的靶基因进行标记，以便更轻松地进行读取。在这里，我们使用了表达靶基因和绿色荧光蛋白（GFP）的双顺反子载体，创建了一个系统，其中 siRNA 序列的作用反映在 GFP 表达水平上。将细胞转导成双顺反子载体、siRNA 表达载体和红色荧光蛋白（RFP）表达载体。转导细胞的流式细胞术分析表明，针对靶基因的 siRNA 沉默了双顺反子载体中的 GFP，但未沉默没有靶基因序列转录的 GFP。此外，RFP 表达细胞上 GFP 的平均荧光强度与靶基因 mRNA 和蛋白水平密切相关。这些结果表明，这种基于流式细胞术的方法使我们能够定量评估 siRNA 的功效和特异性。由于其简单性和有效性，该方法将有助于筛选有效的 siRNA 靶序列，即使在高通量应用中也是如此。
