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Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.
Brett, Sabine I; Lucien, Fabrice; Guo, Charles; Williams, Karla C; Kim, Yohan; Durfee, Paul N; Brinker, C J; Chin, Joseph I; Yang, Jun; Leong, Hon S.
Afiliação
  • Brett SI; Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario.
  • Lucien F; Department of Surgery, Schulich School of Medicine and Dentistry, Western University, London, Ontario.
  • Guo C; Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario.
  • Williams KC; Department of Surgery, Schulich School of Medicine and Dentistry, Western University, London, Ontario.
  • Kim Y; Department of Urology, Mayo Clinic, Rochester, Minnesota.
  • Durfee PN; Department of Mechanical and Materials Engineering, Western University, London, Ontario.
  • Brinker CJ; Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario.
  • Chin JI; Department of Surgery, Schulich School of Medicine and Dentistry, Western University, London, Ontario.
  • Yang J; Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario.
  • Leong HS; Department of Surgery, Schulich School of Medicine and Dentistry, Western University, London, Ontario.
Prostate ; 77(13): 1335-1343, 2017 May.
Article em En | MEDLINE | ID: mdl-28762517
BACKGROUND: The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. METHODS: We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. RESULTS: To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. CONCLUSIONS: While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Próstata / Neoplasias da Próstata / Microscopia de Força Atômica / Vesículas Extracelulares Limite: Humans / Male Idioma: En Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Próstata / Neoplasias da Próstata / Microscopia de Força Atômica / Vesículas Extracelulares Limite: Humans / Male Idioma: En Ano de publicação: 2017 Tipo de documento: Article