# Protocols for Extraction of miRNA from Extracellular Vesicles of Lyophilized Human Saliva Samples

## TL;DR

This paper introduces lyophilization as a new method to preserve and extract microRNAs from saliva extracellular vesicles, offering a scalable alternative to traditional methods.

## Contribution

The study introduces lyophilization as a novel, scalable method for preserving extracellular vesicles and extracting microRNAs from saliva.

## Key findings

- Lyophilized samples retained EV characteristics like CD63/CD9 membrane localization.
- Lyophilized samples showed higher RNA concentrations and preserved key microRNA signatures (miR-16, miR-21, miR-33a, miR-146b).
- Lyophilization effectively reduces solvent content while maintaining vesicle integrity and molecular cargo.

## Abstract

Extracellular vesicles (EVs) are emerging as crucial biomarkers in molecular diagnostics, providing early detection of disease progression. Although ultracentrifugation remains the gold standard for vesicle isolation from biofluids, it has limitations in scalability and accessibility. This study presents lyophilization as an innovative method for preserving EVs and isolating microRNAs from saliva, utilizing its proven ability to maintain biological activity and prevent unwanted chemical reactions. We assessed five different sample preparation protocols combined with a dual-purification strategy. Structural and molecular integrity analyses revealed that lyophilized samples retained essential EV characteristics, including CD63/CD9 membrane localization. QELS analysis and electron microscopy confirmed distinct vesicle populations in both ultracentrifuged (30–50 nm and 320–360 nm) and lyophilized samples (50–70 nm and 360–380 nm). Importantly, lyophilized samples exhibited higher total RNA concentrations (p < 0.0001) while preserving key microRNA signatures (miR-16, miR-21, miR-33a, and miR-146b) with high fidelity. The efficacy of lyophilization is linked to its ability to systematically reduce solvent content through sublimation while maintaining vesicle integrity and molecular cargo. This method offers a practical, scalable alternative for EV isolation with significant implications for biomarker-based diagnostics.