Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Extracellular vesicles (EVs) hold significant clinical potential, but their separation lacks standardization and reproducibility.
• This study addresses the challenge of reliable EV separation from biological samples.

Purpose of the Study:

• To compare the efficacy of three distinct extracellular vesicle (EV) separation protocols.
• To evaluate ultracentrifugation (UC), UC with a purification step (UC+PS), and a polymer-based precipitation combined with UC (PBP+UC) for salivary EV isolation.

Main Methods:

• Salivary samples from healthy donors were processed using UC, UC+PS, and PBP+UC methods.
• EVs were characterized via transmission electron microscopy, nanoparticle tracking analysis, purity assays, RNA quantification, and Western blotting.
• miRNA expression profiling was performed using quantitative RT-PCR.

Main Results:

• All three methods successfully isolated CD9+ and CD63+ EVs from saliva.
• UC+PS and PBP+UC protocols yielded higher EV concentrations, particularly for vesicles under 200 nm.
• EV purity and RNA recovery were comparable across all tested methods, with successful detection of specific miRNAs (miR-16, miR-27a, miR-99a).

Conclusions:

• Both UC+PS and PBP+UC protocols demonstrate comparable efficiency in salivary EV separation.
• The PBP+UC protocol offers a significant advantage due to its simplified processing, making it suitable for initial EV isolation phases.
• The PBP+UC method shows potential for clinical settings requiring rapid and straightforward sample processing, though further validation for transcriptomic and proteomic analyses is recommended.