Abstract
Exosomes are membrane-bound 50–100 nm vesicles released from many cell types including normal and tumorous tissues. Exosomes transport mainly miRNAs, mRNAs, enzymes, cytokines, etc. from the cells of origin to the neighbor cells mediating the communication between them.
The content of exosomes can be explored using RNA profiling after their isolation from medium conditioned by cultured cells or from other biofluids. This chapter includes detailed discussion on isolation, characterization, and miRNA profiling of exosomes. First, exosomes are isolated by filtration and ultracentrifugation, and then characterized using immunoblotting and transmission electron microscope. Finally, we used low density arrays to profile exosomal miRNA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Thery C (2011) Exosomes: secreted vesicles and intercellular communications. F1000 Biol Rep. doi:10.3410/b3-15
Keller S, Sanderson MP, Stoeck A et al (2006) Exosomes: from biogenesis and secretion to biological function. Immunol Lett. doi:10.1016/j.imlet.2006.09.005
Akers JC, Gonda D, Kim R et al (2013) Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol 113:1–11
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
Skog J, Wurdinger T, van Rijn S et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 12:1470–1476
Ginestra A, La Placa MD, Saladino F et al (1998) The amount and proteolytic content of vesicles shed by human cancer cell lines correlates with their in vitro invasiveness. Anticancer Res 18:3433–3437
Clayton A, Mitchell JP, Court J et al (2007) Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res 67:7458–7466
Corcoran C, Rani S, O’Brien K et al (2012) Docetaxel-resistance in prostate cancer: evaluating associated phenotypic changes and potential for resistance transfer via exosomes. PLoS One. doi:10.1371/journal.pone.0050999
O’Brien K, Rani S, Corcoran C et al (2013) Exosomes from triple-negative breast cancer cells can transfer phenotypic traits representing their cells of origin to secondary cells. Eur J Cancer 49:1845–1859
Wang YC, Morrison G, Gillihan R et al (2011) Different mechanisms for resistance to trastuzumab versus lapatinib in HER2-positive breast cancers—role of estrogen receptor and HER2 reactivation. Breast Cancer Res. doi:10.1186/bcr3067
György B, Szabó TG, Pásztói M et al (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68:2667–2688
Kesimer M, Scull M, Brighton B et al (2009) Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense. FASEB J 23:1858–1868
Song J, Bai Z, Han W et al (2012) Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients. Dig Dis Sci 57:897–904
Witwer KW, Buzás EI, Bemis LT, et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. doi:10.3402/jev.v2i0.20360.
Fernández-Llama P, Khositseth S, Gonzales PA et al (2010) Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77:736–742
Rani S, O’Driscoll L (2011) Reverse-transcriptase polymerase chain reaction to detect extracellular mRNAs. Methods Mol Biol. doi:10.1007/978-1-61779-289-2_2
Acknowledgment
This work was supported by funding by Health Research Board of Ireland; Grant number: HRA_POR/2013/341.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Rani, S. (2014). MicroRNA Profiling of Exosomes Isolated from Biofluids and Conditioned Media. In: Alvarez, M., Nourbakhsh, M. (eds) RNA Mapping. Methods in Molecular Biology, vol 1182. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1062-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1062-5_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1061-8
Online ISBN: 978-1-4939-1062-5
eBook Packages: Springer Protocols