Text Box 1. Example of a change from MISEV2014 as a paradigm for considering negative markers.

Argonaute proteins, chiefly AGO2, the most abundant family member in mammals, have been previously proposed as negative markers of EVs. In blood plasma and perhaps other fluids, most extracellular AGO is indeed found outside of EVs [189,190,216]. However, the canonical view of biogenesis of microRNAs (miRNAs) and their subsequent protection from degradation depends on AGO proteins. According to this reasoning, if a mature miRNA is detected, whether in cells, in EVs, or elsewhere, it is most likely protected by AGO (although other associations have been reported [56]). Limits of detection of proteins versus amplified nucleic acids may have contributed to the interpretation that miRNAs are present in EVs without AGO protein. It is in any case now established that AGO can be found in mammalian [217,218] and non-mammalian [219] EVs, not just outside EVs.