# Characterization of RNA in Extracellular Vesicles
## 3 Characterization of RNA in Extracellular Vesicles  
EVs contain various biotypes of RNA that represent a selected portion of the RNA content of the source cell, including noncoding RNA (ncRNA) such as microRNA (miRNA), fragmented and intact mRNA, ribosomal RNA (rRNA), and long noncoding RNA (lncRNA). Several reports have demonstrated that genetic exchange of miRNA or mRNA between cells can be accomplished through microvesicle- or exosome-mediated transfer [34,56]. It was reported that circulating EVs in the plasma are mostly derived from platelets and contain mainly miRNAs, whereby many of these miRNAs are predicted to regulate hematopoiesis and cellular differentiation [57]. Furthermore, endothelial progenitor cell-derived microvesicles activate an angiogenic program in endothelial cells by the horizontal transfer of mRNA [23]. Tumor-derived MVs transfer mRNA to monocytes to activate these cells to produce cytokines, which are involved to enhance tumor growth and dampen the immune response [58]. Accordingly, glioblastoma tumor cells release exosomes containing mRNA, miRNA, but no rRNA, and angiogenic proteins and are able to deliver genetic information to recipient cells in the tumor environment [8]. Additionally, exosomes and special types and sequences of extracellular MV-associated RNAs were useful biomarkers, for example for monitoring prostate cancer invasion and metastasis [8,59,60]. Cellular mechanisms involved in the package of different types of RNA into different types of EVs and their regulated uptake into specific target molecules are still under investigation [61]. Differences in the EV-RNA versus the intracellular RNA profile were described, and it has been suggested that several EV-sorting mechanisms such as different stabilities of special RNA species, which might result from the presence of RNA-binding proteins or special RNases in EVs, must be involved [8,59,62,63,64,65]. Furthermore, special nuclear ribonucleoproteins were suggested to control the sorting of miRNAs into exosomesthrough binding to specific motifs [66]. RNA loading in EVs further depends on cell activation or pathological conditions. For instance, it is well established that stressors such as hypoxia alter the release of EVs and the RNA profile in EVs released in comparison to the donor cells [67]. RNA concentrations in biofluids are very low, 20-50 ng/mL of total plasma, and 5 ng/mL of EV-associated RNA, and several methods have been attempted aiming to quantify and identify different kinds of extracellular RNA in EVs [42; 68]. As expected, the quantity and quality of EV-RNA depend on the method used for EV isolation [40; 43; 69; 70]. Furthermore, the method used for RNA isolation also has an influence on the yield and quality of RNA [42]. In most studies, quantification of isolated RNA is performed using NanoDrop, the Qubit RNA assay, or a bioanalyzer (Agilent), which differ in their detection limit [43]. Only the bioanalyzer, which has the lowest range limit of 50 pg/mL, is able to evaluate size distributions of the RNA molecules additionally. Quantitative real-time polymerase chain reaction (qRT-PCR) is another valuable quantification method [42]. Further molecular analysis of exRNA includes methods such as NanoString, microarray, and next-generation sequencing (NGS) [42]. For further quantitative profiling of small RNAs such as miRNAs, piwit-interacting RNAs (piRNAs), or small nucleolar (sno) RNAs, small RNA sequencing using RNA spike-in molecules is used [71; 72]. So far, most current studies have used microarray analysis and qRT-PCR to examine EV-RNA with a focus on miRNAs. For example, exosomes have been found to contain mainly functional mRNAs and miRNAs [34].