Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper

Abstract

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Towards mechanisms and standardization in extracellular vesicle and extracellular RNA studies: results of a worldwide survey

“…Indeed, the first scientific workshop of the International Society for Extracellular Vesicles (ISEV) was dedicated to the topic of RNA and EVs [9-11]. Since then, ISEV has maintained attention to this topic [12]. In light of the importance of RNA in EV research—but also considering numerous unanswered questions about RNA EV loading, topology, transfer mechanisms, functionality and more—ISEV administered a worldwide survey on EVs and exRNA in 2017.…”

BRAFV600inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

“…The functional role of these small RNAs, especially miRNA, siRNA, and piRNA, is gene silencing by interaction with chromatin or by base pairing with complementary mRNAs or DNAs (6)(7)(8)(9). It has recently been established that RNA molecules not only are retained in the cytoplasm of the cells, but they can also be released into the extracellular milieu, often in extracellular vesicles (EVs) (10,11). It has also been shown that extracellular vesicles can transfer functional RNA between cells (12).…”

Extracellular microRNAs as messengers in the central and peripheral nervous system

“…As much of the research to date has been performed in vitro, it is important to investigate how these identified functions and mechanisms can be translated in vivo. The study of extracellular microRNAs is not without its difficulties [158]. While ultracentrifugation is the most common approach to isolating exosomes, it has its caveats due to the potential negative effects on exosome integrity and the requirement for large volumes of starting material.…”