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Extracellular Vesicles (EVs) gained significant interest within the last decade as a new source of biomarkers for the early detection of diseases and a promising tool for therapeutic applications. In this work, we present Extracellular Vesicles Quantitative Capillary Electrophoresis (EVqCE) to measure an average mass of RNA in EVs, determine EV concentrations and the degree of EV degradation after sample handling. We used EVqCE to analyze EVs isolated from conditioned media of three cancer cell lines. EVqCE employs capillary zone electrophoresis with laser-induced fluorescent detection to separate intact EVs from free nucleic acids. After lysis of EVs with a detergent, the encapsulated nucleic acids are released. Therefore, the initial concentration of intact EVs is calculated based on a nucleic acid peak gain. EVqCE works in a dynamic range of EV concentrations from 108 to 1010 particles/mL. The quantification process can be completed in less than one hour and requires minimum optimization. Furthermore, the average mass of RNA was found to be in the range of 200–400 ag per particle, noting that more aggressive cancer cells have less RNA in EVs (200 ag per particle) than non-aggressive cancer cells (350 ag per particle). EVqCE works well for the degradation analysis of EVs. Sonication for 10 min at 40 kHz caused 85% degradation of EVs, 10 freeze-thaw cycles (from −80 °C to 22 °C) produced 40%, 14-day storage at 4 °C made 32%, and vortexing for 5 min caused 5% degradation. Presently, EVqCE cannot separate and distinguish individual EV populations (exosomes, microvesicles, apoptotic bodies) from each other. Still, it is tolerant to the presence of non-EV particles, protein-lipid complexes, and protein aggregates.

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