Abstract
Circulating microRNAs (miRNAs) have been recently detected in extracellular body fluids and proved themselves as promising biomarkers for a broad spectrum of diseases. The techniques to isolate, detect, and characterize extracellular miRNAs vary significantly from report to report. In this chapter we describe a consistent, efficient and highly reproducible protocol for isolation and detection of microvesicles-free circulating miRNA from human blood plasma or cell culture media. Furthermore, since exosomes-free circulating miRNAs are associated with Argonaut proteins (the same proteins to which miRNAs are bound inside the cell), we provide a protocol for immunoprecipitation of AGO2 associated miRNAs.
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References
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Mitchell PS, Parkin RK, Kroh EM et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105:10513–10518
Hanke M, Hoefig K, Merz H et al (2010) A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer. Urol Oncol 28:655–661
Park NJ, Zhou H, Elashoff D et al (2009) Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin Cancer Res 15:5473–5477
Zubakov D, Boersma AW, Choi Y et al (2010) MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med 124:217–226
Roth C, Rack B, Muller V et al (2010) Circulating microRNAs as blood-based markers for patients with primary and metastatic breast cancer. Breast Cancer Res 12:R90
Gupta SK, Bang C, Thum T (2010) Circulating microRNAs as biomarkers and potential paracrine mediators of cardiovascular disease. Circ Cardiovasc Genet 3:484–488
Wang K, Zhang S, Marzolf B et al (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A 106:4402–4407
Redell JB, Moore AN, Ward NH et al (2010) Human traumatic brain injury alters plasma microRNA levels. J Neurotrauma 27:2147–2156
Kroh EM, Parkin RK, Mitchell PS et al (2010) Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods 50:298–301
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 U S A 108:5003–5008
Turchinovich A, Weiz L, Langheinz A et al (2011) Characterization of extracellular circulating microRNA. Nucleic Acids Res 39:7223–7233
Turchinovich A, Burwinkel B (2012) Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma. RNA Biol 9
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408
Pritchard CC, Cheng HH, Tewari M (2012) MicroRNA profiling: approaches and considerations. Nat Rev Genet 13:358–369
Acknowledgments
This work was supported by Dietmar-Hopp Foundation, the Helmholtz Society, and the German Cancer Research Center. We thank Mrs. Anne Langheinz for helping to establish miRNA isolation protocol.
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© 2013 Springer Science+Business Media New York
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Turchinovich, A., Weiz, L., Burwinkel, B. (2013). Isolation of Circulating MicroRNA Associated with RNA-Binding Protein. 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_8
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DOI: https://doi.org/10.1007/978-1-62703-453-1_8
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-452-4
Online ISBN: 978-1-62703-453-1
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