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Updated: Jul 8, 2025
Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles
Published on: April 6, 2022
First insight into extracellular vesicle-miRNA characterization in a sheep in vitro model of inflammation
Maria Giovanna Ciliberti1, Antonella Santillo1, Agostino Sevi1
1Department of Agriculture, Food, Natural Resources, and Engineering (DAFNE), University of Foggia, Foggia, Italy.
Abstract:
Extracellular vesicles (EVs) and their microRNA (miRNA) cargoes have garnered attention in the veterinary field for their regulatory role in various biological processes. This study aimed to (i) evaluate two techniques of EV isolation from sheep peripheral blood mononuclear cell (PBMC) supernatants using the ultracentrifugation (UC) and reagent (REA) methods and (ii) characterize the EV-miRNA profiles after an in vitro inflammatory environment mediated by lipopolysaccharides (LPS). Sheep peripheral blood was collected, and PBMCs were separated using a density gradient reagent. Subsequently, PBMCs were cultured at 37°C for 24 h (5% CO2), and the supernatants were collected to perform the EV isolation. The presence of CD81+ extracellular vesicle marker was determined, and the purity of isolated EVs was calculated as a ratio between the number of isolated EVs and the protein concentration. Moreover, the morphological characterization revealed mainly round-shaped structures with average sizes of 211 nm for EVs isolated by the UC method and 99 nm for EVs isolated by the REA method. Illumina NextSeq sequencing in a single-end mode was used to characterize the miRNA profile, and the differentially expressed (DE) miRNAs were analyzed using a combination of bioinformatics tools. The results revealed that the REA method is reliable for EV isolation from sheep supernatants. It was considered an improvement of the recovery rate and purity of EVs with the enhancement of the number and the expression levels of characterized miRNAs. The EVs isolated by the UC method after an LPS challenge showed 11 DE miRNAs, among which eight miRNAs were upregulated and three were downregulated. On the other hand, the REA method revealed an EV cargo in which eight DE miRNAs were upregulated and 21 DE miRNAs were downregulated. The master miRNA regulators of the biological process were identified by performing the MIRNA-mRNA network analysis, showing that, among the higher representative miRNAs based on the centrality and betweenness, the miR-26a-5p could have a crucial role in the resolution of inflammation. Moreover, the identification of the let-7 miRNA family in all the EVs showed potential targeted genes that regulate the inflammation and immune responses.
Insights
The reagent method (REA) offers superior isolation of extracellular vesicles (EVs) and their microRNA (miRNA) cargo from sheep blood compared to ultracentrifugation (UC). REA enhances EV purity and miRNA yield, identifying miR-26a-5p as a key regulator in inflammation resolution.
Area of Science:
- Veterinary Science
- Molecular Biology
- Biotechnology
Background:
- Extracellular vesicles (EVs) and their microRNA (miRNA) content are crucial regulators in biological processes, with growing interest in veterinary applications.
- Understanding EV isolation techniques and their miRNA profiles is essential for advancing diagnostic and therapeutic strategies in animal health.
Purpose of the Study:
- To compare ultracentrifugation (UC) and reagent-based (REA) methods for isolating EVs from sheep peripheral blood mononuclear cell (PBMC) supernatants.
- To characterize the miRNA profiles of isolated EVs under *in vitro* inflammatory conditions induced by lipopolysaccharides (LPS).
Main Methods:
- Sheep PBMCs were isolated and cultured, followed by EV isolation using UC and REA methods.
- EV characterization included CD81+ marker detection, purity assessment (EV count/protein concentration), and morphological analysis (size, shape).
- miRNA profiling was performed using Illumina NextSeq sequencing, with differential expression analysis via bioinformatics tools.
Main Results:
- The REA method proved reliable for sheep EV isolation, yielding higher recovery rates, purity, and enhanced miRNA quantity and expression compared to UC.
- UC-isolated EVs showed 11 differentially expressed miRNAs (8 upregulated, 3 downregulated) after LPS challenge.
- REA-isolated EVs exhibited a distinct miRNA profile with 29 differentially expressed miRNAs (8 upregulated, 21 downregulated), highlighting miR-26a-5p's potential role in inflammation resolution and the presence of the let-7 miRNA family.
Conclusions:
- The REA method is a superior technique for isolating high-quality EVs and characterizing their miRNA cargo from sheep PBMC supernatants.
- EV-miRNA profiles differ significantly between UC and REA methods, particularly under inflammatory stimuli.
- miR-26a-5p and the let-7 miRNA family are identified as key regulators of inflammation and immune responses in sheep EVs.
