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Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation

Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods...

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Autores principales: Gutiérrez García, Gilberto, Galicia García, Gabriela, Zalapa Soto, Jessica, Izquierdo Medina, Andrea, Rotzinger-Rodríguez, Mariana, Casas Aguilar, Gustavo Antonio, López Pacheco, Cynthia Paola, Aguayo, Álvaro, Aguilar-Hernandez, Maria Montserrat
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647092/
https://www.ncbi.nlm.nih.gov/pubmed/33156858
http://dx.doi.org/10.1371/journal.pone.0238545
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author Gutiérrez García, Gilberto
Galicia García, Gabriela
Zalapa Soto, Jessica
Izquierdo Medina, Andrea
Rotzinger-Rodríguez, Mariana
Casas Aguilar, Gustavo Antonio
López Pacheco, Cynthia Paola
Aguayo, Álvaro
Aguilar-Hernandez, Maria Montserrat
author_facet Gutiérrez García, Gilberto
Galicia García, Gabriela
Zalapa Soto, Jessica
Izquierdo Medina, Andrea
Rotzinger-Rodríguez, Mariana
Casas Aguilar, Gustavo Antonio
López Pacheco, Cynthia Paola
Aguayo, Álvaro
Aguilar-Hernandez, Maria Montserrat
author_sort Gutiérrez García, Gilberto
collection PubMed
description Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods have surged, such as membrane-affinity column commercial kits. Here, we assessed one commercial kit in terms of EV recovery and EV-derived RNA yield and compared it with a DU protocol. Our data shows that the commercial kit preparation results in a lower count of EV-like structures and a reduced expression of EV markers when compared to DU samples. Thus, apparently suggesting that the commercial kit had a lower EV yield. However, these findings did not reflect on RNA yield, which was greater with the commercial kit, even after an enzymatic treatment with proteinase K and RNAse A. We conclude that the kit has a higher EV-derived RNA yield in comparison to our DU protocol, suggesting that it may be the method of choice for RNA sequencing purposes.
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spelling pubmed-76470922020-11-16 Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation Gutiérrez García, Gilberto Galicia García, Gabriela Zalapa Soto, Jessica Izquierdo Medina, Andrea Rotzinger-Rodríguez, Mariana Casas Aguilar, Gustavo Antonio López Pacheco, Cynthia Paola Aguayo, Álvaro Aguilar-Hernandez, Maria Montserrat PLoS One Research Article Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods have surged, such as membrane-affinity column commercial kits. Here, we assessed one commercial kit in terms of EV recovery and EV-derived RNA yield and compared it with a DU protocol. Our data shows that the commercial kit preparation results in a lower count of EV-like structures and a reduced expression of EV markers when compared to DU samples. Thus, apparently suggesting that the commercial kit had a lower EV yield. However, these findings did not reflect on RNA yield, which was greater with the commercial kit, even after an enzymatic treatment with proteinase K and RNAse A. We conclude that the kit has a higher EV-derived RNA yield in comparison to our DU protocol, suggesting that it may be the method of choice for RNA sequencing purposes. Public Library of Science 2020-11-06 /pmc/articles/PMC7647092/ /pubmed/33156858 http://dx.doi.org/10.1371/journal.pone.0238545 Text en © 2020 Gutiérrez García et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Gutiérrez García, Gilberto
Galicia García, Gabriela
Zalapa Soto, Jessica
Izquierdo Medina, Andrea
Rotzinger-Rodríguez, Mariana
Casas Aguilar, Gustavo Antonio
López Pacheco, Cynthia Paola
Aguayo, Álvaro
Aguilar-Hernandez, Maria Montserrat
Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
title Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
title_full Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
title_fullStr Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
title_full_unstemmed Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
title_short Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
title_sort analysis of rna yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647092/
https://www.ncbi.nlm.nih.gov/pubmed/33156858
http://dx.doi.org/10.1371/journal.pone.0238545
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