Abstract
The isolation and analysis of microRNAs (miRNAs) contained in microvesicles and in particular nano-sized exosomes has become an increasingly important tool to understand their widespread impact in various fundamental and interactive cellular processes. Fundamental studies regarding exosome biogenesis and miRNA sorting may ultimately unravel their potency as a promising class of highly specific disease biomarkers. Here we describe the methods and protocols used in our laboratory to isolate and purify exosomes, how we extract the (small) RNA content, how to analyze copy numbers, and finally how to measure exosome-mediated transfer of these molecules into recipient cells. Our techniques have been optimized for the detection of Epstein–Barr virus (EBV)-encoded miRNAs that are loaded into exosomes. We discuss how a focus on EBV-miRNA detection may yield important new clues into exosome-mediated cross talk by B cells in humans.
Frederik J. Verweij and Monique A.J. van Eijndhoven contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mittelbrunn M, Sánchez-Madrid F (2012) Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol 13:328–335
Yuan A, Farber EL, Rapoport AL et al (2009) Transfer of microRNAs by embryonic stem cell microvesicles. PLoS One 4:e4722
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 9:654–659
Hunter MP, Ismail N, Zhang X et al (2008) Detection of microRNA expression in human peripheral blood microvesicles. PLoS One 3:e3694
Luo S-S, Ishibashi O, Ishikawa G et al (2009) Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes. Biol Reprod 81:717–729
Iorio MV, Visone R, Di Leva G et al (2007) MicroRNA signatures in human ovarian cancer. Cancer Res 67:8699–8707
Kosaka N, Iguchi H, Yoshioka Y et al (2012) Competitive interactions of cancer cells and normal cells via secretory microRNAs. J Biol Chem 287:1397–1405
Sonkoly E, Ståhle M, Pivarcsi A (2008) MicroRNAs and immunity: novel players in the regulation of normal immune function and inflammation. Semin Cancer Biol 18: 131–140
Croce CM, Calin GA (2005) miRNAs, cancer, and stem cell division. Cell 122:6–7
Pegtel DM, Cosmopoulos K, Thorley-Lawson DA et al (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci USA 107:6328–6333
Wurdinger T, Gatson NN, Balaj L et al (2012) Extracellular vesicles and their convergence with viral pathways. Adv Virol 2012:1–12
Pfeffer S, Zavolan M, Grässer FA et al (2004) Identification of virus-encoded microRNAs. Science (New York, NY) 304:734–736
Cosmopoulos K, Pegtel M, Hawkins J et al (2009) Comprehensive profiling of Epstein-Barr virus microRNAs in nasopharyngeal carcinoma. J Virol 83:2357–2367
Qiu J, Cosmopoulos K, Pegtel M et al (2011) A novel persistence associated EBV miRNA expression profile is disrupted in neoplasia. PLoS Pathog 7:e1002193
Kincaid RP, Burke JM, Sullivan CS (2012) RNA virus microRNA that mimics a B-cell oncomiR. Proc Natl Acad Sci USA 109:3077–3082
Feederle R, Haar J, Bernhardt K et al (2011) The members of an Epstein-Barr virus microRNA cluster cooperate to transform B lymphocytes. J Virol 85:9801–9810
Sonkoly E, Ståhle M, Pivarcsi A (2008) MicroRNAs: novel regulators in skin inflammation. Clin Exp Dermatol 33:312–315
Lawrie CH, Saunders NJ, Soneji S et al (2008) MicroRNA expression in lymphocyte development and malignancy. Leukemia 22:1440–1446
Turchinovich A, Weiz L, Langheinz A et al (2011) Characterization of extracellular circulating microRNA. Nucleic Acids Res 39:7223–7233
Arroyo JD, Chevillet JR, Kroh EM et al (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA 108:5003–5008
Gallo A, Tandon M, Alevizos I et al (2012) The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS One 7:e30679
Zomer A, Vendrig T, Hopmans ES et al (2010) Exosomes: fit to deliver small RNA. Commun Integr Biol 3:447–450
Raposo G, Nijman HW, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1161–1172
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Verweij, F.J., van Eijndhoven, M.A.J., Middeldorp, J., Pegtel, D.M. (2013). Analysis of Viral MicroRNA Exchange via Exosomes In Vitro and In Vivo. In: Kosaka, N. (eds) Circulating MicroRNAs. Methods in Molecular Biology, vol 1024. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-453-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-453-1_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-452-4
Online ISBN: 978-1-62703-453-1
eBook Packages: Springer Protocols