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Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

AuthorsLlorens-Revull, Meritxell; Martínez-González, Brenda; Quer, Josep; Esteban, Juan Ignacio; Núñez-Moreno, Gonzalo; Mínguez, Pablo; Burgui, Idoia; Ramos-Ruiz, Ricardo CSIC ORCID; Soria, María Eugenia; Rico, Angie; Riveiro-Barciela, Mar; Sauleda, Silvia; Piron, María; Corrales, Irene; Borràs, Francesc E.; Rodríguez-Frías, Francisco; Rando, Ariadna; Ramírez-Serra, Clara; Camós, Silvia; Domingo, Esteban CSIC ORCID; Bes, Marta; Perales, Celia CSIC ORCID; Costafreda, María Isabel
KeywordsExtracellular vesicle isolation
MiRNA sequencing
Diagnostic biomarker
Issue DateAug-2023
PublisherMultidisciplinary Digital Publishing Institute
CitationInternational Journal of Molecular Sciences 24(15): 12183 (2023)
AbstractMicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.
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