RT Journal Article
T1 Comparative analysis of EV isolation procedures for miRNAs detection in serum samples
A1 Andreu, Zoraida
A1 Rivas, Eva
A1 Sanguino‐Pascual, Aitana
A1 Lamana Domínguez, Amalia
A1 Marazuela, Mónica
A1 González‐Alvaro, Isidoro
A1 Sánchez‐Madrid, Francisco
A1 Fuente, Hortensia de la
A1 Yáñez‐Mó, María
AB 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.
PB Wiley
SN 2001-3078
YR 2016
FD 2016
LK https://hdl.handle.net/20.500.14352/94276
UL https://hdl.handle.net/20.500.14352/94276
LA eng
NO Andreu, Zoraida, et al. «Comparative Analysis of EV Isolation Procedures for miRNAs Detection in Serum Samples». Journal of Extracellular Vesicles, vol. 5, n.o 1, enero de 2016, p. 31655. https://doi.org/10.3402/jev.v5.31655.
NO This work was supported by grant PIE13/00041 from Instituto de Salud Carlos III and co-funded by Fondo Europeo de Desarrollo Regional (FEDER) to MM, IG-A, FS-M and MY-M, and BFU2014-55478-R from Ministerio de Economía y Competitividad to MY-M.
NO Instituto de Salud Carlos III
NO European Commission
NO Ministerio de Economía y Competitividad (España)
DS Docta Complutense
RD 7 abr 2025