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

Abstract：

: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.

journal_name

Mol Ther Methods Clin Dev

journal_title

Molecular therapy. Methods & clinical development

authors

Gaspar LS,Santana MM,Henriques C,Pinto MM,Ribeiro-Rodrigues TM,Girão H,Nobre RJ,Pereira de Almeida L

doi

10.1016/j.omtm.2020.07.012

subject

Has Abstract

pub_date

2020-07-15 00:00:00

pages

723-737

issn

2329-0501

pii

S2329-0501(20)30161-3

journal_volume

18

pub_type

杂志文章

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