Abstract
Exosomes are nano-vesicles present in the circulation that are involved in cell-to-cell communication and regulation of different biological processes. MicroRNAs (miRNAs) are part of their cargo and are potential biomarkers. Methods of exosome isolation and the inter-individual and intra-individual variations in circulating miRNA exosomal cargo have been poorly investigated. This study aims for comparing two exosome isolation methods and to assess the stability of eleven plasma exosomal miRNAs over time. In addition to evaluate miRNA variability of both kits, the effect of freezing plasma before exosome isolation or freezing isolated exosomes on miRNA stability was also evaluated. MiRNA levels were tested in 7 healthy subjects who underwent four different blood extractions obtained in 4 consecutive weeks. One of the isolation kits displayed generally better amplification signals, and miRNAs from exosomes isolated after freezing the plasma had the highest levels. Intra-subject and inter-subject coefficients of variance were lower for the same isolation kit after freezing plasma. Finally, miRNAs that showed an acceptable expression level were stable across the consecutive extractions. This study shows for the first time the stability over time of miRNAs isolated from circulating plasma exosomes, establishing a key step in the use of exosomal miRNAs as biomarkers.
Highlights
Exosomes are double-membrane vesicles with a size between 30 and 150 nm that are formed by the recruitment of the protein Alix to the surface of early endosomes[1]
Dynamic light scattering (DLS) assays showed that isolated vesicles displayed a mean diameter ranging between 43 and 64 nm (Fig. 1A), which was consistent with the exosome population
We evaluated the effect of freezing on the DLS measures and transmission electron microscopy (TEM) images
Summary
Exosomes are double-membrane vesicles with a size between 30 and 150 nm that are formed by the recruitment of the protein Alix to the surface of early endosomes[1]. Exosomes have been isolated from different biofluids, including plasma[9,10], urine[11,12], cerebrospinal fluid[13,14], breast milk[10] and saliva[10] Their widespread localization makes them and their cargo suitable for the development of new biomarkers. MiRNAs are loaded into the exosomes through mechanisms mediated by proteins such as ubiquitin, the Endosomal Sorting Complex Required for Transport (ESCRT) and Y-box protein[25], or miRNA motifs recognized by proteins, such as hnRNPA2B126 This selective packaging explains why exosomes from diseased individuals contain miRNAs different from those found in healthy subjects[27]. Assess the stability of a set of miRNAs involved in inflammation and cardiovascular diseases prior to its application in clinical research
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