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

doi:10.1016/j.isci.2019.100782

Detection of Extracellular Vesicle RNA Using Molecular Beacons

iScience. 2020 Jan 24;23(1):100782. doi: 10.1016/j.isci.2019.100782. Epub 2019 Dec 19.

Affiliations

¹ Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
² Nano Flow Core Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
³ 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.
⁴ Department of Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA, USA.
⁵ Laboratory of Pathology Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
⁶ Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA.
⁷ Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. Electronic address: ighiran@bidmc.harvard.edu.

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.

Keywords: Biochemistry Methods; Biomolecules; Cell Biology; Functional Aspects of Cell Biology.

Detection of Extracellular Vesicle RNA Using Molecular Beacons

Authors

Affiliations

Abstract

Grants and funding