title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.
abstract: Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40-150 nm). The three kits, though, produced a significantly higher yield (80-300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Inas
authors.identifier: http://orcid.org/0000-0002-9294-9277
authors.initials: I
authors.lastname: Helwa
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Cai
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Drewry
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Arthur
authors.initials: A
authors.lastname: Zimmerman
authors.affiliation: Department of Neuroscience and Regenerative Medicine, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Dinkins
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Mariam Lotfy
authors.initials: ML
authors.lastname: Khaled
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Mutsa
authors.initials: M
authors.lastname: Seremwe
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina, United States of America.
authors.forename: W Michael
authors.initials: WM
authors.lastname: Dismuke
authors.affiliation: Department of Neuroscience and Regenerative Medicine, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Erhard
authors.initials: E
authors.lastname: Bieberich
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina, United States of America.|Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Hamrick
authors.affiliation: Department of Cellular Biology and Anatomy, Augusta University, Augusta, Georgia, United States of America.|James & Jean Culver Vision Discovery Institute, Augusta University, Augusta, Georgia, United States of America.|Center for Biotechnology and Genomic Medicine, Augusta University, Augusta, Georgia, United States of America.
authors.forename: Yutao
authors.identifier: http://orcid.org/0000-0003-2870-4504
authors.initials: Y
authors.lastname: Liu
mesh_terms: D015153:Blotting, Western; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles; D016133:Polymerase Chain Reaction / Q000379:methods; D040901:Proteomics; D012313:RNA / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D007202:Indicators and Reagents; D012313:RNA