Application Note – Labeling exosomal RNA and membrane components using fluorescent dyes

Exosomes are small vesicles (30–150 nm) containing sophisticated RNA and protein cargos. They are secreted by all cell types in culture and are found to occur naturally in body fluids, including blood, saliva, urine, CSF, and breast milk [1,2]. The precise molecular mechanics for their secretion and uptake, as well as their composition, “cargo”, and resulting functions, are only beginning to be unraveled. Originally thought to be just “garbage bags” used by cells to get rid of unnecessary macromolecules, exosomes are now viewed as specifically secreted vesicles that enable intercellular communication [2–4]. Exosomes have become the focus of exponentially growing interest, both to study their functions and to understand ways to use them in the development of minimally invasive diagnostics. Critical to furthering our understanding of exosomes is the development of reagents, tools, and protocols for isolation, characterization, and analysis of their RNA and protein content, as well as in vitro and in vivo tracing. A number of techniques may be used to characterize exosomes. These include the use of metabolic incorporation of modified nucleic acids (e.g., ethynyl uridine) or methionine analogs (e.g., homopropargylglycine). Such approaches may allow a more in-depth interrogation of the RNA and protein content of a given vesicle. However, simple protocols for exosomal demarcation and tracking are also very useful. Here we describe protocols for labeling exosomes with two different fluorescent dyes—one selective for RNA and the other for membrane components—that allow researchers to study the pathways and functions of exosomes in cell and animal models.