# Characterization of RNA in Extracellular Vesicles
## 2 Isolation and Characterization of EVs  
Methods employed so far for the isolation of EVs from blood or cell culture supernatants are mostly based on several centrifugation steps, size exclusion chromatography, filtration, precipitation, or the use of magnetic or agarose beads. In most studies, EVs are isolated from the supernatants of cultured cells or from blood samples by performing differential centrifugation steps, whereby at centrifugal forces of 200-1500\(\times\)\(g\), cells and cellular debris of 10,000-20,000\(\times\)\(g\) vesicles with a size between 100 and 800 nm (microvesicles) and between 100,000\(\times\) and 200,000\(\times\)\(g\), vesicles with a diameter <100 nm (exosomes) were pelleted [36]. Additionally, filtration has been used to remove larger vesicles from smaller ones [36]. Furthermore, the presence of characteristic surface proteins on certain EVs is eligible for immunoassay isolation or magnetic activating cell sorting (MACS) [37; 38; 39]. For the isolation of exosomes, the highest yields were produced by using the method of density gradient ultracentrifugation in comparison to the method using differential ultracentrifugation [40]. However, stepped ultracentrifugation procedures cannot achieve absolute separation by size because sedimentation also depends on other parameters such as the type of anticoagulant used, the centrifugal speed to yield platelet-free plasma, the sample viscosity, or the type of rotor used [4; 41]. A variety of further methods to enrich EVs has been described by groups of the Extracellular RNA Communication Consortium, which include methods of density gradients, precipitation, and filtration such as membrane and gel-filtration-based methods. Using syringe filters with successively smaller pore sizes, EVs of different sizes from cell culture supernatants or biofluids were separated, whereas the method of gel filtration or size exclusion chromatography is based on columns containing a solid phase consisting of porous beads that retains smaller molecules whereby larger molecules cannot enter the pores and are eluted from the column quickly [42]. The method of affinity purification, whereby the solid phase is coupled to specific antigens or other molecules present on the surface of EVs, is mostly used to isolate EVs that express disease-specific antigens [4; 43; 42]. Furthermore, a number of commercially available kits were developed to isolate exosomes and MVs, but these kits often fail to distinguish between EVs and other membrane-free macromolecular particles such as protein aggregates [1]. Isolated MVs were characterized by several methods that were reviewed by [PERSON] et al. [4]. Mostly, EVs isolated by differential centrifugation were subsequently studied by flow cytometry [44], whereby scattering flow cytometry requires bead calibration with polystyrene/latex microspheres of known size. However, the detection limit of this method is greater than or equal to 300 nm [45]. Fluorescence-activated cell sorting (FACS) allows classification and specific sorting based on the fluorescence signal of EVs. Although special methods of flow cytometry have been described, the limitation of flow cytometryis its ability to sort EVs <130 nm [5]. The gold-standard method to determine the size of vesicles remains transmission electron microscopy (TEM), which has proven very useful in EV research, as it detects not only the size but also the morphology of isolated vesicles [41]. Furthermore, membrane-surrounded EVs can be recognized and distinguished from other particles such as ribonucleoprotein particles (RNPs) or lipoproteins [1,4]. In general, concentrated EV suspensions are applied to fixed grids, and membrane-surrounded vesicles are recognized by staining with heavy-metal stains such as osmium tetroxide or uranyl acetate [4]. TEM is frequently combined with the use of specific antibodies coupled to nanogold particles for further specification of isolated EVs.