Quantitative Analysis of Extracellular Vesicle Release Using Artificial MicroRNAs

Despite the widely used concept of extracellular vesicle (EV)-mediated intercellular communication, we are still far from understanding what is the exact role of such nanosized vesicles in human physiology and disease. Thus, development of new methods and tools that enable the study of fundamental EV biology is valuable for advancing the field. Typically, EV production and release are monitored using approaches that rely on either antibody-based FACS assays or genetically encoded fluorescent proteins. We previously devised artificially barcoded exosomal microRNAs (bEXOmiRs) that were used as high-throughput reporters of EV release. In the first part of this protocol, basic steps and considerations for the design and cloning of bEXOmiRs are explained in detail. Next, analysis of bEXOmiR expression and abundance in cells and isolated EVs is described.