Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences.
Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences.
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DOI：
10.1016/j.bbrc.2010.02.033
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发表时间：
2010-03-12
影响因子：
3.1
通讯作者：
Imanishi, Jiro
中科院分区：
文献类型：
--
作者：
Kamio, Naoka;Hirai, Hideyo;Ashihara, Eishi;Tenen, Daniel G.;Maekawa, Taira;Imanishi, Jiro
小干扰RNA(SiRNA)的有效性和特异性在很大程度上取决于siRNA序列。由于目前只有经验性的策略可用于预测这些参数，因此需要简单而准确的方法来评估siRNA。为了简化这类实验，为了更容易读出，目标基因经常被标记上记者标签。在这里，我们使用表达目的基因和绿色荧光蛋白(GFP)的双顺反子载体来建立一个系统，其中siRNA序列的影响在GFP表达水平上反映出来。用双顺反子载体、siRNA和红色荧光蛋白(RFP)表达载体转导细胞。流式细胞术分析表明，靶基因的siRNAs沉默了双顺反子载体中的GFP，但不沉默没有靶基因序列的转录的GFP。此外，绿色荧光蛋白在表达RFP的细胞上的平均荧光强度与目的基因的mRNA和蛋白质水平有很好的相关性。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siRNAs的有效性和特异性。由于其简单和有效，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。
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.
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