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The difference between a “leakage” miRNA biomarker and one that is non-randomly packaged into an EV as a putative communication signal, is an important distinction for development of miRNA biomarkers that are indicative of health effects. For example, mammalian miR-122 has been heavily investigated as a leakage biomarker for liver toxicity, in the context of both non-clinical and clinical species. miR-122 accounts for approximately 72% of the total liver miRNAs in mice (Lagos-Quintana et al. 2002) and is highly specific for human, mouse, and rat livers (Landgraf et al. 2007; Smith et al. 2016). In a landmark study by Wang et al. (2009), mice were treated with hepatotoxic levels of acetominophen (APAP) and miRNA microarrays were used to measure difference in the plasma. Forty-four miRNAs were significantly altered, miR-122 showing a 470-fold increase twenty-four hours after APAP overdose, and these alterations could be observed as soon as one hour after exposure. This APAP overdose or acute liver injury-associated miR-122 release was later confirmed in human patients (Starkey Lewis et al. 2011). The observation that decreased liver-based miRNAs with APAP hepatotoxicity correlated with increases in serum plasma suggested that these miRNAs were released as packaged contents or leaked during hepatocyte necrosis; the leakage theory was supported by Arroyo et al. (2011), who demonstrated that miR-122 was primarily associated with a RNP complex (Ago2) and was not membrane-bound as in exosomes. However, later studies provided clues that the mechanism of miR-122 release from liver was context-dependent, that is, dependent on factors such as the condition of the liver, the timing of the injury or perturbation, the half-life of a miRNA, and baseline variability. Subtoxic exposures in vitro result primarily in hepatocyte-derived release of exosome-bound miR-122 (Holman et al. 2016; Mosedale et al. 2018), and alcoholic and inflammatory liver disease was associated with exosome-bound miR-122—as opposed to drug-induced injury, which was found in the protein-bound fraction of plasma (Bala et al. 2012). Further, the protection of miRNA from RNases present in biofluids may preferentially enrich exosome and protein-bound miRNAs (Arroyo et al. 2011; Koberle et al. 2013; Li et al. 2012; Turchinovich et al. 2011).

Therefore the decision whether to quantify miRNA biomarkers from unfractionated samples or from isolated EVs may depend on context and on intended use. Additionally, questions remain as to which one is more reliable in biomarker measurements; and some reports suggest that this may depend on the specific human disease. A disease-specific alteration in exosome release and clearance, or the administration of therapeutics that alter the volume of blood, can skew the yield (Buschmann et al. 2018; Hanna et al. 2019). The isolation of miRNAs from unfractionated biofluid samples should capture both miRNAs that originate from passive leakage into the sampled matrix and miRNAs that come from actively released membrane-bound vesicles lysed through the isolation procedure. This isolation avoids major drawbacks of vesicle purification, which lowers RNA yield and integrity and can result is greater experimental variability. Another challenge is that, depending on the method used, different populations of EVs are isolated; and this produces only partially overlapping RNA profiles (Buschmann et al. 2018). Recently, a comprehensive analysis of published results compared the utility of miRNAs isolated from EVs to the utitliy of those isolated from unfractionated whole serum or plasma for specific biomarker use (Nik Mohamed Kamal and Shahidan 2019). Twenty of the thirty-two studies cited suggested that EV-derived miRNAs may be preferable for the intended use examined. A commonly reported reason was the potential for increased stability that might be derived from being encapsulated in a vesicle. But it is also important to note that these intended uses varied, and included both in vitro and in vivo biomarker development for heart failure, chronic pain, septic shock, non-alcoholic fatty liver disease, immunization, and many types of cancer. So, while a slight majority of studies recommended vesicle fraction miRNA for biomarker measurement, specific research needs to be performed to provide the appropriate conditions and context of use.