title: Reproducibility and efficiency of serum-derived exosome extraction methods.
abstract: OBJECTIVE
  Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum.
  
  
  METHODS
  We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94.
  
  
  RESULTS
  Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG.
  
  
  CONCLUSIONS
  ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.
authors: Vancouver Prostate Centre, University of British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Canada; Faculty of Medicine, University of British Columbia, Canada. Electronic address: jcaradec@prostatecentre.com.
authors: Josselin
authors: J
authors: Caradec
authors: Vancouver Prostate Centre, University of British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Canada; Faculty of Medicine, University of British Columbia, Canada.
authors: Geetanjali
authors: G
authors: Kharmate
authors: Vancouver Prostate Centre, University of British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Canada; Faculty of Medicine, University of British Columbia, Canada.
authors: Elham
authors: E
authors: Hosseini-Beheshti
authors: Vancouver Prostate Centre, University of British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Canada; Faculty of Medicine, University of British Columbia, Canada.
authors: Hans
authors: H
authors: Adomat
authors: Vancouver Prostate Centre, University of British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Canada; Faculty of Medicine, University of British Columbia, Canada.
authors: Martin
authors: M
authors: Gleave
authors: Vancouver Prostate Centre, University of British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Canada; Faculty of Medicine, University of British Columbia, Canada.
authors: Emma
authors: E
authors: Guns
mesh_terms: D000418:Albumins / Q000378:metabolism; D015415:Biomarkers / Q000097:blood*; D055354:Exosomes / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D052119:Lysosomal-Associated Membrane Protein 2 / Q000097:blood; D008562:Membrane Glycoproteins / Q000097:blood; D053758:Nanoparticles / Q000737:chemistry; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D060245:Tetraspanin 29 / Q000097:blood; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D000418:Albumins; D015415:Biomarkers; C554869:CD9 protein, human; D007074:Immunoglobulin G; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D008562:Membrane Glycoproteins; D060245:Tetraspanin 29; C053387:endoplasmin
keywords: ExoQuick™; Exosomes; Serum; Standardisation; Ultracentrifugation