Abstract
Urinary exosomes are nanovesicles (40–100 nm) of endocytic origin that are secreted into the urine when a multivesicular body fuses with the membrane of cells from all nephron segments. Interest in urinary exosomes intensified after the discovery that they contain not only protein and mRNA but also microRNA (miRNA) markers of renal dysfunction and structural injury. Currently, the most widely used protocol for the isolation of urinary exosomes is based on ultracentrifugation, a method that is time consuming, requires expensive equipment, and has low scalability, which limits its applicability in the clinical practice.
In this chapter, a simple, fast, and highly scalable step-by-step method for isolation of urinary exosomes is described. This method starts with a 10-min centrifugation of 10 ml urine, then the supernatant is saved (SN1), and the pellet is treated with dithiothreitol and heat to release and recover those exosomes entrapped by polymeric Tamm–Horsfall protein. The treated pellet is then resuspended and centrifuged, and the supernatant obtained (SN2) is combined with the first supernatant, SN1. Next, 3.3 ml of ExoQuick-TC, a commercial exosome precipitation reagent, is added to the total supernatant (SN1 + SN2), mixed well, and saved for at least 12 h at 4 °C. Finally, a pellet of exosomes is obtained after a 30-min centrifugation of the supernatant/ExoQuick-TC mix. We previously compared this method with five others used to isolate urinary exosomes and found that this is the simplest, fastest, and most effective alternative to ultracentrifugation-based protocols if the goal of the study is RNA profiling. A method for isolation and quantification of miRNAs and mRNAs from urinary exosomes is also described here. In addition, we provide a step-by-step description of exosomal miRNA profiling using universal reverse transcription and SYBR qPCR.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hewitt SM, Dear J, Star RA (2004) Discovery of protein biomarkers for renal diseases. J Am Soc Nephrol 15:1677–1689
Tomlanovich S, Golbetz H, Perlroth M et al (1986) Limitations of creatinine in quantifying the severity of cyclosporine-induced chronic nephropathy. Am J Kidney Dis 8:332–337
Tsalamandris C, Allen TJ, Gilbert RE (1994) Progressive decline in renal function in diabetic patients with and without albuminuria. Diabetes 43:649–655
Macisaac RJ, Jerums G (2011) Diabetic kidney disease with and without albuminuria. Curr Opin Nephrol Hypertens 20:246–257
Pisitkun T, Johnstone R, Knepper MA (2006) Discovery of urinary biomarkers. Mol Cell Proteomics 5:1760–1771
Nilsson J, Skog J, Nordstrand A et al (2009) Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br J Cancer 100:1603–1607
Van Balkom BW, Pisitkun T, Verhaar MC et al (2011) Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases. Kidney Int 80:1138–1145
Miranda KC, Bond DT, McKee M et al (2010) Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int 78:191–199
Valadi H, Ekstrom 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
Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101:13368–13373
Zhou H, Yuen PS, Pisitkun T et al (2006) Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int 69:1471–1476
Gonzales PA, Pisitkun T, Hoffert JD et al (2009) Large-scale proteomics and phosphoproteomics of urinary exosomes. J Am Soc Nephrol 20:363–379
Gonzales PA, Zhou H, Pisitkun T et al (2010) Isolation and purification of exosomes in urine. Methods Mol Biol 641:89–99
Fernandez-Llama P, Khositseth S, Gonzales PA et al (2010) Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77:736–747
Thery C, Clayton A, Amigorena S et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 3:22.1–22.29
Cheruvanky A, Zhou H, Pisitkun T et al (2007) Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol 292:F1657–F1661
Taylor DD, Zacharias W, Gercel-Taylor C (2011) Exosome isolation for proteomic analyses and RNA profiling. Methods Mol Biol 728:235–246
Alvarez ML, Khosroheidari M, Kanchi Ravi R et al (2012) Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int 82:1024–1032
Kato M, Zhang J, Wang M et al (2007) MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors. Proc Natl Acad Sci U S A 104:3432–3437
Putta S, Lanting L, Sun G et al (2012) Inhibiting microRNA-192 ameliorates renal fibrosis in diabetic nephropathy. J Am Soc Nephrol 23:458–469
Alvarez ML, Khosroheidari M, Eddy E et al (2013) Role of microRNA 1207-5p and its host gene, the long non-coding RNA PVT1, as mediators of extracellular matrix accumulation in the kidney: implications for diabetic nephropathy. PLoS One 8:e77468
Alvarez ML, DiStefano JK (2011) Functional characterization of the plasmacytoma variant translocation 1 gene (PVT1) in diabetic nephropathy. PLoS One 6:e18671
Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, research0034
Chen C, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179
Gautier JC, Riefke B, Walter J et al (2010) Evaluation of novel biomarkers of nephrotoxicity in two strains of rat treated with Cisplatin. Toxicol Pathol 38:943–956
Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5520
Andreasen D, Fog JU, Biggs W et al (2010) Improved microRNA quantification in total RNA from clinical samples. Methods 50:S6–S9
Castoldi M, Schmidt S, Benes V et al (2008) miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes. Nat Protoc 3:321–329
Beuvink I, Kolb FA, Budach W et al (2007) A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs. Nucleic Acids Res 35:e52
McAlexander MA, Phillips MJ, Witwer KW (2013) Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from cerebrospinal fluid. Front Genet 4:83
Mestdagh P, Van Vlierberghe P, De Weer A et al (2009) A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 10:R64
Chang KH, Mestdagh P, Vandesompele J et al (2010) MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer. BMC Cancer 10:173
Pfaffl MW, Tichopad A, Prgomet C et al (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515
Acknowledgements
The author is grateful to the Roney Family Foundation for the support of this study, to Madieh (Tala) Khosroheidari and Dr. Rupesh Kanchi Ravi for their valuable help in performing part of the experiments, and to Paul Arnold for the critical review of this manuscript.
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
Alvarez, M.L. (2014). Isolation of Urinary Exosomes for RNA Biomarker Discovery Using a Simple, Fast, and Highly Scalable Method. 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_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1062-5_13
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