Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research

Detalhes bibliográficos
Autor(a) principal: Gaspar, Laetitia da Silva
Data de Publicação: 2020
Outros Autores: Santana, Magda M., Henriques, Carina, Pinto, Maria M., Ribeiro-Rodrigues, Teresa M., Girão, Henrique, Nobre, Rui Jorge, Almeida, Luís Pereira de
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10316/106458
https://doi.org/10.1016/j.omtm.2020.07.012
Resumo: Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 109 ± 1.22 × 108 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 1010 ± 7.38 × 109 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice.
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spelling Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Researchbiomarkersclinical researchextracellular vesiclesplasmasize exclusion chromatographytherapytranscriptional researchExtracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 109 ± 1.22 × 108 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 1010 ± 7.38 × 109 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice.This work was co-financed by the European Joint Programme Neurodegenerative Disease Research (JPND) and Fundação para a Ciência e a Tecnologia (FCT), under the projects European Spinocerebellar Ataxia Type 3/Machado-Joseph Disease Initiative (ESMI, JPCOFUND/ 0001/2015, 01/BIM-ESMI/2016), ModelPolyQ (JPCOFUND/ 0005/2015) and SynSpread; the European Regional Development Fund (ERDF) through the Operational Programme for Competitiveness and Internationalisation - COMPETE 2020 and Portuguese national funds via FCT, under the projects Brain- Health2020 (CENTRO-01-0145-FEDER-000008), ViraVector (CENTRO- 01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/ 0084/2014 and POCI-01-0145-FEDER-016719), SpreadSilencing (POCI-01-0145-FEDER-029716), noOSAnoAGEING (POCI-01- 0145-FEDER-029002), Imagene (POCI-01-0145-FEDER-016807), CancelStem (POCI-01-0145-FEDER-016390, POCI-01-0145-FEDER- 032309, and UIDB/04539/2020); the National Ataxia Foundation (USA); AFMTelephon; the American Portuguese Biomedical Research Fund (APBRF); the Richard Chin and Lily LockMachado-Joseph Disease Research Fund; and the European Social Fund through the Human Capital Operational Programme (POCH) and Portuguese national funds via FCT, under PD/BD/135497/2018.Elsevier2020-09-11info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/106458http://hdl.handle.net/10316/106458https://doi.org/10.1016/j.omtm.2020.07.012eng2329-0501Gaspar, Laetitia da SilvaSantana, Magda M.Henriques, CarinaPinto, Maria M.Ribeiro-Rodrigues, Teresa M.Girão, HenriqueNobre, Rui JorgeAlmeida, Luís Pereira deinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-04-05T13:55:27Zoai:estudogeral.uc.pt:10316/106458Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:22:54.556120Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
title Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
spellingShingle Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
Gaspar, Laetitia da Silva
biomarkers
clinical research
extracellular vesicles
plasma
size exclusion chromatography
therapy
transcriptional research
title_short Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
title_full Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
title_fullStr Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
title_full_unstemmed Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
title_sort Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research
author Gaspar, Laetitia da Silva
author_facet Gaspar, Laetitia da Silva
Santana, Magda M.
Henriques, Carina
Pinto, Maria M.
Ribeiro-Rodrigues, Teresa M.
Girão, Henrique
Nobre, Rui Jorge
Almeida, Luís Pereira de
author_role author
author2 Santana, Magda M.
Henriques, Carina
Pinto, Maria M.
Ribeiro-Rodrigues, Teresa M.
Girão, Henrique
Nobre, Rui Jorge
Almeida, Luís Pereira de
author2_role author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Gaspar, Laetitia da Silva
Santana, Magda M.
Henriques, Carina
Pinto, Maria M.
Ribeiro-Rodrigues, Teresa M.
Girão, Henrique
Nobre, Rui Jorge
Almeida, Luís Pereira de
dc.subject.por.fl_str_mv biomarkers
clinical research
extracellular vesicles
plasma
size exclusion chromatography
therapy
transcriptional research
topic biomarkers
clinical research
extracellular vesicles
plasma
size exclusion chromatography
therapy
transcriptional research
description Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 109 ± 1.22 × 108 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 1010 ± 7.38 × 109 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice.
publishDate 2020
dc.date.none.fl_str_mv 2020-09-11
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10316/106458
http://hdl.handle.net/10316/106458
https://doi.org/10.1016/j.omtm.2020.07.012
url http://hdl.handle.net/10316/106458
https://doi.org/10.1016/j.omtm.2020.07.012
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 2329-0501
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
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dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
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