Author(s): 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
Date: 2020
Persistent ID: https://hdl.handle.net/10316/106458
Origin: Estudo Geral - Universidade de Coimbra
Subject(s): biomarkers; clinical research; extracellular vesicles; plasma; size exclusion chromatography; therapy; transcriptional research
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.
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.
