Advertisement

Protocols for Exosome Isolation and RNA Profiling

  • Fernando Marqués-GarcíaEmail author
  • María Isidoro-García
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1434)

Abstract

Exosomes are small extracellular vesicles of multivesicular bodies derived from the cell endosome. Molecules have a characteristic composition both in the membrane and in the carried load. These particles are located in the culture medium of cells in vitro and in vivo body fluids, being synthesized by almost all kind of cells. The most characteristic molecule transported within is the RNA with a large clinical potential. In this chapter, the methodology for isolation of exosomes and subsequent purification of the RNA is reviewed.

Key words

Biomarkers Exosome Extracellular vesicles Isolation Precipitation RNA profiling Ultracentrifugation 

References

  1. 1.
    Mathivanan S, Ji H Simpson RJ et al (2010) Exosome: extracellular organelles important in intercellular communication. J Proteomics 73(10):1907–1920Google Scholar
  2. 2.
    Lee Y, EL Andaloussi S, Wood MJA (2012) Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Hum Mol Genet 21(R1):R125–R134CrossRefPubMedGoogle Scholar
  3. 3.
    Blanchard N, Lankar D, Faure F et al (2002) TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex. J Immunol 168:3235–3241CrossRefPubMedGoogle Scholar
  4. 4.
    Raposo G, Nijman HW, Stoorvoge W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183(3):1161–1172CrossRefPubMedGoogle Scholar
  5. 5.
    Thery C, Regnault A, Garin J et al (1999) Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73. J Cell Biol 147(3):599–610CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wolfers J, Lozier A, Raposo G et al (2001) Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 7(3):297–3037CrossRefPubMedGoogle Scholar
  7. 7.
    Waldestrom A, Gennebäck N, Hellman U et al (2012) Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PLoS One 7(4):e34653CrossRefGoogle Scholar
  8. 8.
    Ronquist G (2012) Proteasomes are mediators of intercellular communication: from basic research to clinical implications. J Intern Med 271(4):400–413CrossRefPubMedGoogle Scholar
  9. 9.
    Choi DS, Kim DK, Kim YK et al (2014) Proteomics of extracellular vesicles: exosomes and ectosomes. Mass Spectrom Rev 34(4):474–490CrossRefPubMedGoogle Scholar
  10. 10.
    Caby MPD, Vincendeau-Scherrer C et al (2005) Exosomal-like vesicles are present in human blood plasma. Int Immunol 17(7):879–887CrossRefPubMedGoogle Scholar
  11. 11.
    Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101(36):13368–13373CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Admyre C, Johansson SM, Qazi KR et al (2007) Exosomes with immune modulatory features are present in human breast milk. J Immunol 179(3):1969–1978CrossRefPubMedGoogle Scholar
  13. 13.
    Admyre C, Grunewald J, Thyberg J et al (2003) Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid. Eur Respir J 22(4):578–583CrossRefPubMedGoogle Scholar
  14. 14.
    Vlassov AV, Magdaleno S, Setterquist R et al (2012) Exosomes: Current knowledge of their composition, biological functions, and diagnostics and therapeutic potentials. Biochim Biophys Acta 1820(7):940–948Google Scholar
  15. 15.
    Al-Nedawi K, Meehan B, Micallef J et al (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10(5):619–624CrossRefPubMedGoogle Scholar
  16. 16.
    Szeles A, Henriksson M et al (2001) Horizontal transfer of oncogenes by uptake of apoptotic bodies. Proc Natl Acad Sci U S A 98(11):6407–6411CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Holmgren L, Szeles A, Rajnavölgyi E et al (1999) Horizontal transfer of DNA by the uptake of apoptotic bodies. Blood 93:3956–3963PubMedGoogle Scholar
  18. 18.
    Lösche W, Scholz T, Temmler U et al (2004) Platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils. Platelets 15(2):109–115CrossRefPubMedGoogle Scholar
  19. 19.
    Schageman J, Zeringer E, Mu Li et al (2013) The complete exosome workflow solution: from isolation to characterization of RNA cargo. BioMed Res Int 2013:15 pGoogle Scholar
  20. 20.
    Théry C, Zitvogel L, Amigorena S (2002) Exosome: composition, biogénesis and function. Nat Rev Immunol 2:569–579PubMedGoogle Scholar
  21. 21.
    Janowska-Wieczorek A, Wysoczynski M, Kijowski J et al (2005) Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer. Int J Cancer 113:752–760CrossRefPubMedGoogle Scholar
  22. 22.
    Lakkaraju A, Rodriguez-Boulan E (2008) Itinerant exosomes: emerging roles in cell and tissue polarity. Trends Cell Biol 18(5):199–209CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Rajendran L, Honsho M, Zahn TR et al (2006) Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc Natl Acad Sci U S A 103:11172–11177CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Peinado H, Alečković M, Lavotshkin S et al (2012) Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 18:883–891CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Admyre C, Telemo E, Almqvist N et al (2008) Exosomes—nanovesicles with possible roles in allergic inflammation. Allergy 63:404–408CrossRefPubMedGoogle Scholar
  26. 26.
    Suntres EZ, Smith MG, Momen-Heraviv F et al (2013) Therapeutic uses of exosomes. Exosomes Microvesicles 1:2013Google Scholar
  27. 27.
    Valadi H, Ekström K, Bossios A et al (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659CrossRefPubMedGoogle Scholar
  28. 28.
    Huang X, Yuan T, Tschannen M et al (2013) Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 14:319CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Mathivanan S, Simpson RJ (2009) ExoCarta: a compendium of exosomal proteins and RNA. Proteomics 9:4997–5000CrossRefPubMedGoogle Scholar
  30. 30.
    Kim DK, Kang B, Kim OY et al (2013) EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles. J Extracell Vesicles 2:20384CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Fernando Marqués-García
    • 1
    • 2
    Email author
  • María Isidoro-García
    • 1
    • 2
    • 3
  1. 1.Department of Clinical BiochemistryUniversity Hospital of SalamancaSalamancaSpain
  2. 2.Salamanca Institute for Biomedical Research (IBSAL)SalamancaSpain
  3. 3.Department of MedicineUniversity of SalamancaSalamancaSpain

Personalised recommendations