双荧光蛋白报告基因分析系统在微小RNA研究中的应用

The application of dual fluorescent protein reporter assay system in microRNA research

目的:建立双荧光蛋白报告基因分析系统,并用来验证微小RNA的靶基因。方法:选取表达绿色荧光蛋白的质粒pcDNA3/EGFP,将待验证微小RNA靶基因的一段特异性序列插入该质粒中,并与表达红色荧光蛋白质粒pDsRed2-N1共同转染细胞,转染后的细胞和提取的蛋白样品,分别用荧光显微镜和荧光分光光度计进行定性和定量检测。结果:通过对一特定小RNA(miR-27a)的可能靶基因prohibitin进行验证,最终确定prohibitin是miR-27a的靶基因。结论:双荧光蛋白报告基因分析系统是一种验证微小RNA靶基因的可靠方法。

Objective： To establish dual fluorescent protein reporter assay system, and identify target gene of microRNA by using this method. Methods： We inserted a specific sequence of candidated target gene into the plasmid in which green fluorescent protein had expressed （pcDNA3/EGFP）. The plasmid and the red fluorescent protein plasmid （pDsRed2 -N1 ） were cotransfected into ceils. The cells and the extracted protein were detected by the fluorescence microscope and the fluorescence spectrophotometer respectively. Results： This research demonstrated that prohibitin was a target gene of miR-27a with using this assay system. Conclusion： Dual fluorescent protein reporter assay system is a reliable method by which target gene of microRNA can be identified.