Abstract
Extracellular vesicles (EVs) are cell-secreted, lipid membrane-enclosed nanoparticles without functional nucleus. EV is a general term that includes various subtypes of particles named microvesicles, microparticles, ectosomes or exosomes. EVs transfer RNA, DNA and protein cargo between proximal and distant cells and tissues, thus constituting an organism-wide signal transduction network. Pathological tissues secrete EVs that differ in their cargo composition compared to their healthy counterparts. The detection of biomarkers in EVs from biological fluids may aid the diagnosis of disease and/or monitor its progression in a minimally invasive manner. Among biological fluids, pleural effusions (PEs) are integrated to clinical practice, as they accompany a wide variety of lung disorders. Due to the proximity with the pleura and the lungs, PEs are expected to be especially enriched in EVs that originate from diseased tissues. However, PEs are among the least studied biofluids regarding EV-specialized isolation methods and related biomarkers. Herein, we describe a practical EV isolation method from PEs for the screening of EV RNA biomarkers in clinical routine. It is based on a Proteinase K treatment step to digest contaminants prior to standard polyethylene-glycol precipitation. The efficiency of the method was confirmed by transmission electron microscopy, nanoparticle tracking analysis and Western blot. The reliability and sensitivity of the method towards the detection of EV-enriched RNA biomarkers from multiple PEs was also demonstrated.
Highlights
Introduction published maps and institutional affilPleural effusion (PE) is the excessive accumulation of fluid in the pleural space
The Extracellular vesicles (EVs) suspensions were lysed by the addition of RIPA and the concentration of sEV proteins was determined by the bicinchoninic acid (BCA) assay (Thermo Fisher Inc., Rockford, IL, USA) using bovine serum albumin (BSA) as standard. sEV positive marker
PEs are clinical manifestations of diseases that have advanced to their later stages, and they represent a relevant biofluid for vesicle-based liquid biopsy analyses
Summary
Two studies have optimized the parameters of sEV isolation by PEG precipitation from cell cultured media [30,31]. The efficiency of sEV isolation increases in proportion to the molecular weight and the final concentration of the PEG solution added, as well as the incubation time after the addition of PEG. The maximum efficiency is achieved by the addition of PEG ranging from 6000 to 8000 kDa in solutions to a final concentration of. PEG precipitation is the more efficient method. The method is based on two previous studies [30,31] and uses conditions to ensure maximum efficiency of EV isolation. K digestion step in our method to counteract the inherently low purity of PEG precipitation and the abundance of contaminants in the PE.
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