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Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing
MicroRNAs (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...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10418926/ https://www.ncbi.nlm.nih.gov/pubmed/37569568 http://dx.doi.org/10.3390/ijms241512183 |
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author | Llorens-Revull, Meritxell Martínez-González, Brenda Quer, Josep Esteban, Juan Ignacio Núñez-Moreno, Gonzalo Mínguez, Pablo Burgui, Idoia Ramos-Ruíz, Ricardo 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 Bes, Marta Perales, Celia Costafreda, Maria Isabel |
author_facet | Llorens-Revull, Meritxell Martínez-González, Brenda Quer, Josep Esteban, Juan Ignacio Núñez-Moreno, Gonzalo Mínguez, Pablo Burgui, Idoia Ramos-Ruíz, Ricardo 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 Bes, Marta Perales, Celia Costafreda, Maria Isabel |
author_sort | Llorens-Revull, Meritxell |
collection | PubMed |
description | MicroRNAs (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. |
format | Online Article Text |
id | pubmed-10418926 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-104189262023-08-12 Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing Llorens-Revull, Meritxell Martínez-González, Brenda Quer, Josep Esteban, Juan Ignacio Núñez-Moreno, Gonzalo Mínguez, Pablo Burgui, Idoia Ramos-Ruíz, Ricardo 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 Bes, Marta Perales, Celia Costafreda, Maria Isabel Int J Mol Sci Article MicroRNAs (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. MDPI 2023-07-29 /pmc/articles/PMC10418926/ /pubmed/37569568 http://dx.doi.org/10.3390/ijms241512183 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Llorens-Revull, Meritxell Martínez-González, Brenda Quer, Josep Esteban, Juan Ignacio Núñez-Moreno, Gonzalo Mínguez, Pablo Burgui, Idoia Ramos-Ruíz, Ricardo 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 Bes, Marta Perales, Celia Costafreda, Maria Isabel Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing |
title | Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing |
title_full | Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing |
title_fullStr | Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing |
title_full_unstemmed | Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing |
title_short | Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing |
title_sort | comparison of extracellular vesicle isolation methods for mirna sequencing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10418926/ https://www.ncbi.nlm.nih.gov/pubmed/37569568 http://dx.doi.org/10.3390/ijms241512183 |
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