Generation and Characterization of In Vitro Transcribed mRNA

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.

Keywords: Flow cytometry; Fluorescence spectrophotometry; Immunofluorescence microscopy; In vitro transcription; Intracellular staining; LNPs; Lipid nanoparticles; Ultraviolet spectrophotometry; Western blotting; mRNA capping.