Detection of Extracellular Vesicle RNA Using Molecular Beacons

Abstract

Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.

Technologies and Standardization in Research on Extracellular Vesicles

“…In the area of biochemical characterization of EVs, enabling the following capabilities appears especially attractive: detailed molecular cataloging of EV populations of various properties, origins, and functions; sensitive quantitative characterization of PTMs of EV proteins (e.g., glycosylation, acetylation, phosphorylation); informative multiomics characterization of the same EV isolates from the same specimen; high-sensitivity deep molecular profiling of EV isolates from limited Trends Trends in in Biotechnology Biotechnology samples (finger-prick blood specimens, neonatal specimens, tear fluid; see the above-listed examples); and highly specific and sensitive detection and quantitation of target EV molecules of interest [205] in EV isolates, nonprocessed physiological fluids (e.g., whole blood, CSF), minimally processed biological fluids (e.g., plasma, serum), and even archived samples derived from physiological fluids (e.g., dried blood spots, small aliquots of longitudinally collected cohort studies) that can enable the detection and quantitation of rare EVs in the bulk of other, irrelevant EV populations at high sensitivity and specificity.…”

Urinary Biomarkers in Bladder Cancer: Where Do We Stand and Potential Role of Extracellular Vesicles

“…To fulfill such requirements, innovative optical [ 179 , 180 , 181 ], nano-flow cytometry [ 182 , 183 , 184 , 185 ], Raman [ 164 , 186 , 187 ] and other plasmonic sensors methods [ 188 , 189 , 190 , 191 ] have recently emerged for highly sensitive single-EV detection. Nevertheless, their application has not been applied to urine samples and the analysis of single-EVs and other submicron particles has presented many challenges and has produced a few controversial results in other types of samples.…”

Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

“…For the detection of EVs using CytoFLEX, the sample flow rate was adjusted to slow (10 µL/min) [ 24 , 25 ]. The stop criterion was set for a time at 300 s or the events 10,000 [ 26 ]. The data were analyzed using CytExpert (Beckman Coulter, Brea, CA, USA).…”