Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-b...

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Published inOral diseases
Main Authors Itzel, Castillejos-García, Eduardo, Martínez-Martínez, Velia, Ramírez-Amador, Mireya, Cisneros-Villanueva, Alfredo, Hidalgo-Miranda, Pilar, Ramos-Godínez María Del, Gabriela, Anaya-Saavedra
Format Journal Article
LanguageEnglish
Published Denmark 27.10.2024
Subjects
saliva
PEG
extracellular vesicles
miRNAs
ultracentrifugation
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Summary:The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC). Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA. All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods. The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.
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ISSN:1354-523X
1601-0825
1601-0825
DOI:10.1111/odi.15171