Simultaneous and multiplexed detection of exosome microRNAs using molecular beacons

Simultaneous and multiplexed detection of microRNAs (miRNAs) in a whole exosome is developed, which can be utilized as a PCR-free efficient diagnosis method for various diseases. Exosomes are small extracellular vesicles that contain biomarker miRNAs from parental cells. Because they circulate throughout bodily fluids, exosomal biomarkers offer great advantages for diagnosis in many aspects. In general, PCR-based methods can be used for exosomal miRNA detection but they are laborious, expensive, and time-consuming, which make them unsuitable for high-throughput diagnosis of diseases. Previously, we reported that single miRNA in the exosomes can be detected specifically using an oligonucleotide probe or molecular beacon. Herein, we demonstrate for the first time that multiple miRNAs can be detected simultaneously in exosomes using miRNA-targeting molecular beacons. Exosomes from a breast cancer cell line, MCF-7, were used for the production of exosomes because MCF-7 has a high level of miR-21, miR-375, and miR-27a as target miRNAs. Molecular beacons successfully hybridized with multiple miRNAs in the cancer cell-derived exosomes even in the presence of high human serum concentration. In addition, it is noteworthy that the choice of fluorophores for multiplexing biomarkers in an exosome is crucial because of its small size. The proposed method described in this article is beneficial to high-throughput analysis for disease diagnosis, prognosis, and response to treatment because it is a time-, labor-, and cost-saving technique.