ISEV2025 Abstract Book
Résumé
Introduction: Extracellular vesicles are lipid-bound nanopar- ticles that play a crucial role in cell-to-cell communication, influencing both physiological and pathological processes. Their presence in peripheral biofluids, including saliva, an easily acces- sible and non-invasive sample source, makes them promising candidates for biomarker discovery in diagnostics and prog- nostics. Additionally, microRNAs (miRNAs) are increasingly recognized as valuable diagnostic tools for various diseases. Although multiple methods exist for isolating and characterizing salivary EVs (sEVs), further optimization and standardization are necessary to facilitate their clinical translation. This study aims to characterize and compare the miRNA content of sali- vary EVs isolated using two rapid and scalable techniques, co-precipitation (Q) and immunoaffinity (M), against the gold standard ultracentrifugation (UC) method. Methods: Saliva samples were collected from three healthy volunteers. sEVs were isolated using UC, Q, and M. Following a comprehensive characterization, miRNAs were extracted from both sEVs and whole saliva (WS) and subsequently sequenced using next-generation sequencing (NGS). Results: Our analysis revealed that while saliva and EVs share common sets of miRNAs, several miRNAs exhibit differential expression between WS and sEVs. Principal component analysis demonstrated a clear separation between WS and the three isolation methods. UC and Q showed a strong similarity, cluster- ing primarily by individual rather than by isolation technique, whereas M exhibited a separation pattern driven more by the isolation method than by individual variation. Despite these differences, all three methods yielded highly consistent miRNA enrichment profiles when compared to WS. Gene Ontology and KEGG pathway analyses indicated significant enrichment of miRNA-related processes, including small RNA pathways and splicing regulation, particularly in the M condition. Summary/Conclusion: This study highlights the significance of sEVs as a source of miRNA and demonstrates that certain miRNA species are preferentially enriched in WS, while others are predominantly contained within sEVs. The three isolation techniques provided robust and consistent miRNA profiles, with UC and Q exhibiting strong similarities and M showing superior enrichment in specific pathways. These findings underscore the importance of EV isolation before miRNA analysis and confirm that all three methods are suitable for clinical salivary sample processing