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Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples

Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and u...

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Autores principales: Royo, Felix, Zuñiga-Garcia, Patricia, Sanchez-Mosquera, Pilar, Egia, Ainara, Perez, Amparo, Loizaga, Ana, Arceo, Raquel, Lacasa, Isabel, Rabade, Ainara, Arrieta, Edurne, Bilbao, Roberto, Unda, Miguel, Carracedo, Arkaitz, Falcon-Perez, Juan M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Co-Action Publishing 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759834/
https://www.ncbi.nlm.nih.gov/pubmed/26895490
http://dx.doi.org/10.3402/jev.v5.29497
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author Royo, Felix
Zuñiga-Garcia, Patricia
Sanchez-Mosquera, Pilar
Egia, Ainara
Perez, Amparo
Loizaga, Ana
Arceo, Raquel
Lacasa, Isabel
Rabade, Ainara
Arrieta, Edurne
Bilbao, Roberto
Unda, Miguel
Carracedo, Arkaitz
Falcon-Perez, Juan M.
author_facet Royo, Felix
Zuñiga-Garcia, Patricia
Sanchez-Mosquera, Pilar
Egia, Ainara
Perez, Amparo
Loizaga, Ana
Arceo, Raquel
Lacasa, Isabel
Rabade, Ainara
Arrieta, Edurne
Bilbao, Roberto
Unda, Miguel
Carracedo, Arkaitz
Falcon-Perez, Juan M.
author_sort Royo, Felix
collection PubMed
description Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs.
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spelling pubmed-47598342016-03-09 Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples Royo, Felix Zuñiga-Garcia, Patricia Sanchez-Mosquera, Pilar Egia, Ainara Perez, Amparo Loizaga, Ana Arceo, Raquel Lacasa, Isabel Rabade, Ainara Arrieta, Edurne Bilbao, Roberto Unda, Miguel Carracedo, Arkaitz Falcon-Perez, Juan M. J Extracell Vesicles Original Research Article Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs. Co-Action Publishing 2016-02-15 /pmc/articles/PMC4759834/ /pubmed/26895490 http://dx.doi.org/10.3402/jev.v5.29497 Text en © 2016 Felix Royo et al. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Research Article
Royo, Felix
Zuñiga-Garcia, Patricia
Sanchez-Mosquera, Pilar
Egia, Ainara
Perez, Amparo
Loizaga, Ana
Arceo, Raquel
Lacasa, Isabel
Rabade, Ainara
Arrieta, Edurne
Bilbao, Roberto
Unda, Miguel
Carracedo, Arkaitz
Falcon-Perez, Juan M.
Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
title Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
title_full Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
title_fullStr Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
title_full_unstemmed Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
title_short Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
title_sort different ev enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples
topic Original Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759834/
https://www.ncbi.nlm.nih.gov/pubmed/26895490
http://dx.doi.org/10.3402/jev.v5.29497
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