Abstract
Small extracellular vesicles are released from both healthy and disease cells to facilitate cellular communication. They have a wide variety of names including exosomes, microvesicles and microparticles. Depending on their size, very small extracellular vesicles originating from the endocytic pathway have been called exosomes and in some cases nanovesicles. Collectively, extracellular vesicles are important mediators of a wide variety of functions including immune cell development and homeostasis. Encapsulated in the extracellular vesicles are proteins and nucleic acids including mRNA and microRNA molecules. MicroRNAs are small, non-coding RNA molecules implicated in the post-transcriptional control of gene expression that have emerged as important regulatory molecules and are involved in disease pathogenesis including cancer. In some diseases, not only does the quantity and the subpopulations of extracellular vesicles change in the peripheral blood but also microRNAs. Here, we described the analysis of peripheral blood extracellular vesicles by flow cytometry and the RNA extraction from extracellular vesicles isolated from the plasma or serum to profile microRNA expression.
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References
Hunter MP, Ismail N, Zhang X et al (2008) Detection of microRNA expression in human peripheral blood microvesicles. PLoS One 3:e3694
Rozmyslowicz T, Majka M, Kijowski J et al (2003) Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV. AIDS 17:33–42
Nieuwland R, Berckmans RJ, McGregor S et al (2000) Cellular origin and procoagulant properties of microparticles in meningococcal sepsis. Blood 95:930–935
Valenti R, Huber V, Iero M et al (2007) Tumor-released microvesicles as vehicles of immunosuppression. Cancer Res 67:2912–2915
Taylor DD, Gercel-Taylor C (2011) Exosomes/microvesicles: mediators of cancer-associated immunosuppressive microenvironments. Semin Immunopathol 33:441–454. doi:10.1007/s00281-010-0234-8
Taylor DD, Gercel-Taylor C (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 110:13–21
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 10:1470–1476
Rabinowits G, Gercel-Taylor C, Day JM et al (2009) Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer 10:42–46
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 113:752–760
Iero M, Valenti R, Huber V et al (2008) Tumour-released exosomes and their implications in cancer immunity. Cell Death Differ 15:80–88. doi:4402237
Hong BS, Cho JH, Kim H et al (2009) Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells. BMC Genomics 10:556–568
Mitchell PS, Parkin RK, Kroh EM et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105:10513–10518
Thery C, Boussac M, Veron P et al (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166:7309–7318
Shet AS, Aras O, Gupta K et al (2003) Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 102:2678–2683. doi:10.1182/blood-2003-03-0693
Wang G, Lai K-B, Kwa BC-H et al (2012) Non-vesicle-bound free microRNAs could enter cells and affect gene expression. Adv Biosci Biotechnol 3:181–185. doi:10.4236/abb.2012.33026
Jiang J, Lee EJ, Piper MG et al (2011) High-throughput profiling of mature microRNA by real-time PCR. In: Harper SQ (ed) RNA interference techniques, vol 58. Humana Press, New York, NY, pp 113–121. doi:10.1007/978-1-61779-114-7
Jayachandran M, Miller VM, Heit JA et al (2012) Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J Immunol Methods 375:207–214. doi:S0022-1759
Willems M, Moshage H, Nevens F et al (1993) Plasma collected from heparinized blood is not suitable for HCV-RNA detection by conventional RT-PCR assay. J Virol Methods 42:127–130
Al-Soud WA, Radstrom P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39:485–493. doi:10.1128/JCM.39.2.485-493.2001
Calzuola I, Gianfranceschi GL, Marsili V (2001) Binding citrate/DNA in presence of divalent cations. Potential mimicry of acidic peptides/DNA interactions. Mol Biol Rep 28:43–46
Ismail N, Wang Y, Dakhlallah D et al (2013) Macrophage Microvesicles Induce Macrophage Differentiation and miR-223 Transfer. Blood 121:984–995
Acknowledgements
The authors would like to acknowledge the ongoing collaboration and assistance of Drs. Thomas Schmittgen and Jinmai Jiang (The Ohio State University), in developing and modifying the qRT-PCR protocol to profile miRNA expression from the extracellular vesicles.
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Moldovan, L., Batte, K., Wang, Y., Wisler, J., Piper, M. (2013). Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR. 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_10
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DOI: https://doi.org/10.1007/978-1-62703-453-1_10
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Publisher Name: Humana Press, Totowa, NJ
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