Inside the Cell: Approaches to Evaluating mRNA Internalization and Trafficking

This is especially important when working with novel mRNA constructs, comparing different delivery vectors, or targeting diverse cell types. In this study, we present a suite of methods to quantify and visualize mRNA internalization following transfection of three types of human primary cells: esenchymal stromal cells, fibroblasts, and osteoblasts.

We highlight the utility of fluorescent probes for both qualitative and quantitative assessment of mRNA uptake and intracellular trafficking. To dissect the pathways involved in uptake, we employed three distinct endocytic inhibitors— chlorpromazine, wortmannin, and genistein—each targeting specific endocytic mechanisms. Additionally, we provide protocols for the lipid-based transfection agents Lipofectamine 3000 and 3DFect, which can be adapted for use with similar vectors.