Stability of Circulating Exosomal miRNAs in Healthy Subjects

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.

Human Pluripotent Stem Cell-Derived Extracellular Vesicles: Characteristics and Applications

“…The EV stability over time for miRNAs (e.g., miR-16, 21, 126, 143, 145, 150, 222, and 320) isolated from circulating plasma was evaluated, establishing a key step in the use of exosomal miRNAs as biomarkers. 58 The plasma can be frozen before EV/exosome isolation or isolated EVs/exosomes can be later frozen to ensure miRNA stability. The storage temperature was found to be critical for EV stability (over 25 days) and a range of -20°C to -80°C was required to maintain exosomal marker expression.…”

The Therapeutic Potential of Breast Milk-Derived Extracellular Vesicles

“…Innovations in exosome biology have allowed researchers to engineer exosomes/EVs for delivery of specific miRNA cargo [49,50]. Exosomes and miRNAs are highly stable and can withstand the stresses of the GI tract and the bloodstream, strengthening their case as a potentially tunable therapeutic system [51][52][53]. This can be done through transfection of a parent cell with an miRNA-mimic to over-produce a particular miRNA, or by transfecting exosomes/EVs directly, leading to an increase in miRNAs packaged within exosomes [35,49,50,54].…”

Epigenetics dysfunction in morbid obesity with or without obstructive sleep apnoea: the EPIMOOSA study

“…Circulating exosomes and the encapsulated miRNA will be studied at the Translational Unit of the Miguel Servet Hospital in Zaragoza. Exosomes will be isolated with the miRCURY™Exosome Isolation Kit (Qiagen, Venlo, Netherlands), and miRNA will be obtained using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Venlo, Netherlands) as previously described [32]. After extracting the RNA samples, they will be reverse transcribed using the miRCURY LNA™ Universal RT miRNA PCR Kit (Qiagen, Venlo, Netherlands).…”

“…Mature miRNA will then be quantified by real-time quantitative PCR using PCR Master Mix (Qiagen, Venlo, Netherlands). The integrity of the analyses will be checked using the recommended spike-in control as previously described [32]. The results will be expressed following the 2 -ΔΔ threshold cycle (Ct) method [33].…”