Extracellular vesicles (EVs) gained significant interest within the last decade as a new source of biomarkers for the early detection of diseases and as a promising tool for therapeutic applications. As a result, a need for new methods for EV analysis and quantification has elevated. In this work, I apply Extracellular Vesicles Quantitative Capillary Electrophoresis (EVqCE) to determine (i) the apparent molecular weight of RNA in EVs, (ii) the number of intact EVs in a sample, and (iii) the degree of EV degradation after sonication, vortexing, freeze-thaw cycles and long storage. This separation method is demonstrated on EVs isolated from conditioned media of three different cancer cell lines and human urine samples. Here, I utilize capillary zone electrophoresis with laser-induced fluorescent detection to separate intact EVs from DNA and RNA impurities present in the sample. YOYO-1 dye is used to stain all DNA and RNA in the sample. After lysis of EVs with a detergent, encapsulated DNA and RNA are released. After additional RNase treatment of the EVs sample, RNA is enzymatically cleaved, leaving residual DNA only, in order to calculate the RNA concentration from EVs. Therefore, the initial concentration of intact EV is calculated based on the gain of a nucleic acid peak in capillary electrophoresis and an RNA calibration curve. 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 for CE separation.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/41015 |
| Date | 16 September 2020 |
| Creators | Dou, Yuchu |
| Contributors | Berezovski, Maxim |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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