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.
Keywords: RNA biomarkers; exosomes; extracellular vesicles; pleural effusion; proteinase K.