title: Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.
abstract: Extracellular vesicles (EVs) are emerging as potent non-invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV-encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation-based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR-126, miR-30c and miR-143), while the detection of miR-21, miR-16-5p and miR-19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut-off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.
authors.affiliation: Unidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Zoraida
authors.initials: Z
authors.lastname: Andreu
authors.affiliation: Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Rivas
authors.affiliation: Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Aitana
authors.initials: A
authors.lastname: Sanguino-Pascual
authors.affiliation: Servicio de Reumatología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Amalia
authors.initials: A
authors.lastname: Lamana
authors.affiliation: Servicio de Endocrinología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Marazuela
authors.affiliation: Servicio de Reumatología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Isidoro
authors.initials: I
authors.lastname: González-Alvaro
authors.affiliation: Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Madrid
authors.affiliation: Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain; hortensia.fuente@salud.madrid.org.
authors.forename: Hortensia
authors.initials: H
authors.lastname: de la Fuente
authors.affiliation: Unidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.|Departamento de Biología Molecular, UAM/CBM-SO, Madrid, Spain; maria.yanez@salud.madrid.org; maria.yannez@uam.es.
authors.forename: María
authors.initials: M
authors.lastname: Yáñez-Mó
keywords: biomarker; diagnosis; extracellular vesicles; microRNA; polyethylene glycol; serum