# Characterization of RNA in Extracellular Vesicles
## 3 Characterization of RNA in Extracellular Vesicles
Because unknown miRNAs or other RNA species are often undetectable, a sequencing-based RNA profiling analysis from exosomes isolated from blood samples demonstrated that plasma-derived exosomes not only contain miRNA and mRNA but also a large variety of other small noncoding RNA species, including RNA transcript, repeat sequences, structural RNAs, and tRNA fragments [73; 74; 75]. Furthermore, deep sequencing or RNA in exosomes from the colon cancer cell line identified special miRNA sequences enriched in EVs compared to the parent cancer cell [76]. In future, the use of deep sequencing will ensure the characterization of the whole transcriptome of blood-derived exosomes. However, the distribution of different kinds of RNA in EVs is highly dependent on the method or kit used for the isolation of EVs, and the co-precipitation of non-EV microparticles or RNA-binding proteins should be excluded [74]. Size filtration of conditioned medium from glioblastoma stem-like cell cultures reveals that mRNA is enriched in MVs, whereas exosomes have the highest fraction of miRNA [42; 72]. Using the method of differential centrifugation, the RNA profile showed that rRNA was preferred detectable in apoptotic bodies and smaller RNAs, but no rRNAs were present in exosomes. In these studies, detection, quality, yield, and size of vesicular RNAs were analyzed using capillary electrophoresis by the bioanalyzer (Agilent) [48]. Accordingly, rRNA and additionally DNA are mainly present in microparticles from cells undergoing apoptosis in vitro [77; 78]. Table 1 summarizes types of RNA isolated from different EV populations. Type of RNA in EVs is furthermore dependent on the source used for EV isolation. Subpopulations of EVs isolated from metastatic tumor tissue showed distinct size and morphology and differed in their RNA cargo [79]. Type of RNA identified in different EVs isolated from cell culture supernatants was not only dependent on the type of donor cell but also on the type of stimulation [48; 80]. After culturing the mast cell line HMC-1 in normal growth medium, MVs isolated from supernatants did not contain rRNA, but when HMC-1 was treated with degranulating agents such as ionomycin or complement factor in serum-free medium, MVs contained high amounts of rRNA and fewer small RNAs [48; 80]. Other studies confirmed that the type of RNA, present in EVs isolated from cell-culture-conditioned medium, is dependent on culture conditions. For example, bovine serum also contains RNA in EVs, and methods to remove exRNA from bovine serum through ultracentrifugation or affinity purification are only partly successful so far [81; 82]. In summary, many studies failed to demonstrate whether identified extracellular RNAs were associated with EVs or rather with RNA-protein complexes, viral particles, RNPs, and lipoproteins (HDL and LDL) that might have been co-isolated with EVs [1, 43]. However, in this regard, it has been reported that EVs isolated by ultracentrifugation may become aggregated and are contaminated by nonvesicular macromolecules. Additional purification techniques such as high-resolution density gradient fractionation and direct immunoaffinity capture techniques were used to characterize EVs and nonvesicular material precisely [83]. DNA known to be present in several biological fluids was also demonstrated to be present in EV-RNA preparations and thus can interfere with the downstream analysis of RNA [84]. rRNA might also become associated with EVs during centrifugation steps. This might explain the variable presence of rRNA in EVs described in the literature, and the question of whether rRNAs are naturally associated with EVs or if they are impurities or contaminants is still to be discussed [43]. To remove outside bound DNA and RNA and destroy protein complexes such as RNPs, EVs that will be used for the isolation of RNA have to be pretreated with DNase, RNase, and proteinase [43].