Comparison of exosomes purified via ultracentrifugation (UC) and Total Exosome lsolation (TEI) reagent from the serum of Marek's disease virus (MDV)-vaccinated and tumor bearing chickens

Extracellular vesicles (EVs) is a collective term used to refer microparticles, exosomes, and apoptotic bodies produced by a variety of cells and released into interstitial spaces and bodily fluids. Serum exosomes can serve as invaluable biomarkers, containing m/miRNAs, lipids, and proteins, indicative of various conditions. There are currently limited studies on the characterization and mutual consensus of biomarker profiles of serum exosomes purified by different methods. Here we compared the advantages and disadvantages of two commonly used serum exosome purification procedures including ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent, by analyzing exosome size distribution, concentration, morphology and miRNA expression profiles. Serum was obtained from Marek's disease virus (MDV)-infected chickens that were either vaccinated against Marek's disease (MD), and thus protected, or unvaccinated and bearing MDV-induced tumors. Nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy (TEM) were performed to evaluate particle size, concentration, and morphological integrity, respectively. Our results indicate that the size distribution of particles purified by either procedure is consistent with that of exosomes (30-150 nm). TEI reagent generated higher yields and co-isolated additional EV populations that are slightly larger (similar to 180 nm). Based on the miRNA expression profiles from a previous high throughput sequencing experiment of exosome small RNAs, we selected six cellular and four MDV1 miRNAs, to validate their expression in UC- and TEI-purified exosomes. miRNA expression profiles displayed relative correlation between the two procedures, but distinctive differences were observed in abundance with TEI-purified exosomes showing higher miRNA expression consistent with higher yield than those purified by UC. TEI-purified exosomes from vaccinated chickens exhibited greater expression of tumor suppressor miRNA, gga-mir-146b and least expression of oncomiR, gga-mir-21 compared to those obtained from tumor-bearing chickens. We propose that gga-mir.146 and -21 can serve as serum exosome biomarkers for vaccine-induced protection and MD tumors respectively.