Abstract：
Here we describe the in vitro preparation of mRNA from DNA templates, including setting up the transcription reaction, mRNA capping, and mRNA labeling. We then describe methods used for mRNA characterization, including UV and fluorescence spectrophotometry, as well as gel electrophoresis. Moreover, characterization of the in vitro transcribed RNA using the Bioanalyzer instrument is described, allowing a higher resolution analysis of the target molecules. For the in vitro testing of the mRNA molecules, we include protocols for the transfection of various primary cell cultures and the confirmation of translation by intracellular staining and western blotting.
摘要：
在这里，我们描述了从DNA模板体外制备mRNA，包括建立转录反应，mRNA加帽，和mRNA标记。然后我们描述用于mRNA表征的方法，包括紫外和荧光分光光度法，以及凝胶电泳。此外，描述了使用Bioanalyzer仪器对体外转录RNA的表征，允许对目标分子进行更高分辨率的分析。对于mRNA分子的体外测试，我们包括各种原代细胞培养物的转染和通过细胞内染色和蛋白质印迹确认翻译的方案。
