Comparison of Methods for the Isolation of Salivary Extracellular Vesicles.

Extracellular vesicles (EVs) have attracted growing attention for their diagnostic and prognostic potential as they carry molecular cargo such as DNA, RNA, proteins and lipids derived from their cells of origin. While EV research has traditionally focused on blood, this study explicitly explored saliva as a promising, non-invasive sample matrix for EV isolation and biomarker discovery. Six different EV isolation methods were compared for their ability to recover salivary small EVs suitable for downstream DNA and microRNA analysis. Nanoparticle tracking analysis (NTA) revealed variation in vesicle sizes, concentrations and surface charges across all tested EV isolation approaches. In addition to being the fastest and simplest isolation method, the miRCURY Exosome Isolation kit-serum and plasma from Qiagen (ExiQ) also resulted in the highest EV yields with average particle sizes of ~130 nm. Western blot analysis further verified the presence of EV-specific markers (CD9, Alix) and no detectable signal for ApoA1 as an indicator for lipoprotein contamination, underscoring the purity of ExiQ-isolated vesicles. Always applying the same protocol for parallel DNA and RNA isolation on vesicles extracted by various methods, differences in DNA and RNA yields were observed across the evaluated isolation kits. ExiQ-isolated EVs showed the best recovery for both nucleic acid types. Notably, nuclease treatment of isolated EVs revealed that substantial amounts of DNA were present on the EV surface, whereas microRNA was predominantly localized within the vesicles. The present study, extensively comparing different EV isolation methods, demonstrates that salivary EVs are a viable source for non-invasive diagnostics and suggests the miRCURY Exosome Isolation kit-serum and plasma from Qiagen (ExiQ) to be a good choice for integration in future salivary EV-based diagnostic assays given its simplicity, speed and excellent performance.