Detection of Extracellular Vesicle RNA Using Molecular Beacons
Getulio Pereira de Oliveira, Jr.,
Eric Zigon,
Gaenna Rogers,
Danny Davodian,
Shulin Lu,
Tijana Jovanovic-Talisman,
Jennifer Jones,
John Tigges,
Sanjay Tyagi,
Ionita Calin Ghiran
Affiliations
Getulio Pereira de Oliveira, Jr.
Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Eric Zigon
Nano Flow Core Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Gaenna Rogers
Nano Flow Core Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Danny Davodian
Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; Centre for Prehospital and Emergency Research, Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
Shulin Lu
Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Tijana Jovanovic-Talisman
Department of Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA, USA
Jennifer Jones
Laboratory of Pathology Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
John Tigges
Nano Flow Core Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Sanjay Tyagi
Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA
Ionita Calin Ghiran
Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; Corresponding author
Summary: 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. : Biochemistry Methods; Biomolecules; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Biochemistry Methods, Biomolecules, Cell Biology, Functional Aspects of Cell Biology
