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 × 10 9 ± 1.22 × 10 8 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 × 10 10 ± 7.38 × 10 9 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.

Extracellular vesicles as predictors of individual response to exercise training in youth living with obesity

“…As per MISEV guidelines, we first measured the purity of isolated EV fractions. We showed an increase in the expression of proteins enriched in exosomes, TSG101 and CD81 in F7–10 as reported previously (32, 33). While F10 was not completely free of contamination by lipoproteins (ApoA1) and non-exosomal proteins (Cyt C), it also showed the presence of sEVs (~56.3 nm, data not shown).…”

“…To validate SEC-based EV isolation in our study, as per MISEV guidelines (29) we analyzed each fraction (F1–12) for protein yield and expression of exosome-specific (TSG101 and CD81) or non-exosomal markers or negative controls (Cyt C and Apo-A1). Protein concentration increased nearly exponentially starting in F7 through to F12 ( Figure 1B ) as shown before (32, 33). Exosomal markers TSG101 and CD81 were enriched in F7–10.…”

Adsorption of extracellular vesicles onto the tube walls during storage in solution

“…Precleared ovarian follicular fluids might be kept at +4˚C for up to a week prior to EVs isolation [38]. The storage at +4˚C of purified EVs for up to 48 hours is allowed by protocol [39] (isolation by size-exclusion chromatography) and for 72 hours by protocol [40] (isolation by ultracentrifugation), while [41] recommend keeping isolated EVs on ice and process them as soon as possible. This ambiguity indicates the necessity of careful examination of the EVs instability phenomenon at +4˚C.…”

“…No direct experimental evidence for this route was reported for EVs in peer-reviewed papers, but multiple researchers warned that this process might occur during storage [6,20,33,42]. Some studies and protocols reported the usage of low protein binding tubes for EVs storage or handling [32,41,[43][44][45] with no experimental basis for this choice. Recently a patent for hydrophilic polymeric coating/additive to prevent EV adsorption was published [46] together with commercial EV-Save™ blocking reagent (058-09261, FUJIFILM Wako Pure Chemical Corporation).…”

SLC6A8 Knockdown Suppresses the Invasion and Migration of Human Hepatocellular Carcinoma Huh-7 and Hep3B Cells

“…The concentration of extracted protein was determined by BCA assay. 17 Proteins were separated using 12% SDS-polyacrylamide gel electrophoresis before transferred to polyvinylidene fluoride (PVDF) membranes using a wet membrane apparatus and blocked for 1 h with PBS containing 5% skimmed milk powder. 18 Details of primary antibodies used are in Table 3 .…”