Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma

Abstract

Background Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application. Methods In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods. Results The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value. Conclusions SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.

Establishment of a simplified dichotomic size‐exclusion chromatography for isolating extracellular vesicles toward clinical applications

“…Brennan et al showed that the qEV for HS EV isolation could obtain the particle/protein ratios ranged 10 8 ∼10 9 particles/μg protein (Brennan et al., 2020 ). Yang et al performed regular SEC on HS, and they observed the particle/protein ratios to be ranged from 7 × 10 8 ∼1 × 10 9 particles/μg protein (Yang et al., 2021 ). Takov et al found that the particle/protein ratio of ∼1.9 × 10 8 particles/μg protein could be achieved from the EV isolation from rat plasma using the qEVorigenal SEC columns (Takov et al., 2019 ).…”

Asymmetric depth-filtration – a versatile and scalable approach for isolation and purification of extracellular vesicles

“…The yield of EVs per mL of human plasma was significantly higher by depth filtration (Figure 2c). Therefore, out of the three examined isolation methods, the depth filtration is the least biased 8,14,57 , providing the most accurate representation of extracellular vesicles present in the blood. EV biomarkers (CD63, CD81, CD9, and EpCAM) were most expressed in DF-isolated samples.…”

The Potential Application of Extracellular Vesicles from Liquid Biopsies for Determination of Pharmacogene Expression

“…Isolation of EVs can be performed based on size, weight or their compositio mon methods for EV isolation are ultracentrifugation (UC), size-exclusion chr raphy (SEC), precipitation, immunoaffinity and filtration methods which can be combination (Figure 2) [22].…”

“…The isolation and characterization methods are often chosen in line with the specific sample and goal. The most important factors in choosing the method of isolation are yield and purity [22].…”

“…Isolation of EVs can be performed based on size, weight or their composition. Common methods for EV isolation are ultracentrifugation (UC), size-exclusion chromatography (SEC), precipitation, immunoaffinity and filtration methods which can be used in combination (Figure 2) [22]. Immunoaffinity methods require an antibody (for example for CD9, CD81 or CD63) conjugated with beads which are upon binding with EVs separated magnetically.…”