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Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle Capture from Plasma Samples

  • Davide ZoccoEmail author
  • Natasa Zarovni
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1660)

Abstract

Extracellular vesicle (EV)-associated RNAs (EV-RNA) are under intense investigation due to their potential role in health and disease. Several approaches are currently employed to isolate blood-derived EVs for RNA analysis, most of which are either time-consuming and expensive, such as methods based on EVs physical properties (ultracentrifugation and Optiprep density gradient), or also copurify blood contaminants, mostly protein aggregates and immune complexes, (such as chemical precipitation). In addition, there is a lack of standardized protocols for the extraction of EV-RNA and very little consensus on the technological platforms and normalization tools for assessing the expression levels of different RNA species. These methodological issues complicate the comparison between independent data sets, potentially biasing results and conclusions.

In this book chapter we propose a protocol that might overcome some of the abovementioned issues through antibody-based isolation of blood-derived EVs followed by extraction and expression analysis of small-RNA species (miRNA) by reverse transcriptase quantitative PCR (RT-qPCR). The advantages of immunoaffinity approaches over other isolation methods are multiple and include: (1) the selective enrichment of specific EV subpopulations with restricted tissue/cell origin, (2) reduction of matrix effects and blood contaminants that may confound miRNA profiling from complex biological fluids and (3) easy coupling to conventional quantitative assays (e.g., RT-qPCR). In conclusion, we describe a protocol for standard enrichment and quantitative analysis of EV-miRNAs from blood and we warrant for technological improvements, such as the use of novel biomaterials, surface chemistries, binding agents and assay/sensor design that may further improve it.

Key words

Extracellular vesicles miRNA Antibody-coated beads Plasma RT-qPCR 

References

  1. 1.
    Rak J (2013) Extracellular vesicles – biomarkers and effectors of the cellular interactome in cancer. Front Pharmacol 4:21CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Yuana Y, Sturk A, Nieuwland R (2013) Extracellular vesicles in physiological and pathological conditions. Blood Rev 27(1):31–39CrossRefPubMedGoogle Scholar
  3. 3.
    Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, Molina H, Kohsaka S, Di Giannatale A, Ceder S, Singh S, Williams C, Soplop N, Uryu K, Pharmer L, King T, Bojmar L, Davies AE, Ararso Y, Zhang T, Zhang H, Hernandez J, Weiss JM, Dumont-Cole VD, Kramer K, Wexler LH, Narendran A, Schwartz GK, Healey JH, Sandstrom P, Labori KJ, Kure EH, Grandgenett PM, Hollingsworth MA, de Sousa M, Kaur S, Jain M, Mallya K, Batra SK, Jarnagin WR, Brady MS, Fodstad O, Muller V, Pantel K, Minn AJ, Bissell MJ, Garcia BA, Kang Y, Rajasekhar VK, Ghajar CM, Matei I, Peinado H, Bromberg J, Lyden D (2015) Tumour exosome integrins determine organotropic metastasis. Nature 527(7578):329–335CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zocco D, Ferruzzi P, Cappello F, Kuo WP, Fais S (2014) Extracellular vesicles as shuttles of tumor biomarkers and anti-tumor drugs. Front Oncol 4:267CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wang K, Yuan Y, Cho JH, McClarty S, Baxter D, Galas DJ (2012) Comparing the MicroRNA spectrum between serum and plasma. PLoS One 7(7):e41561CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Witwer KW, Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, Nolte-’t Hoen EN, Piper MG, Sivaraman S, Skog J, Théry C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in EV research. J Extracell Vesicles 2Google Scholar
  7. 7.
    Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A (2014) The impact of disparate isolation methods for EV on downstream RNA profiling. J Extracell Vesicles 3Google Scholar
  8. 8.
    Chevillet JR, Kang Q, Ruf IK, Briggs HA, Vojtech LN, Hughes SM, Cheng HH, Arroyo JD, Meredith EK, Gallichotte EN, Pogosova-Agadjanyan EL, Morrissey C, Stirewalt DL, Hladik F, Yu EY, Higano CS, Tewari M (2014) Quantitative and stoichiometric analysis of the microRNA content of exosomes. PNAS 41:4888–14893Google Scholar
  9. 9.
    Turchinovich A, Weiz L, Langheinz A, Burwinkel B (2011) Characterization of extracellular circulating microRNA. Nucleic Acids Res 39(16):7223–7233CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhang J, Li S, Li L, Li M, Guo C, Yao J, Mi S (2015) Exosomes and exosoman microRNAs: trafficking, sorting and function. Genomics Proteomics Bioinformatics 13(1):17–24CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Grasedieck S, Sorrentino A, Langer C, Buske C, Döhner H, Mertens D, Kuchenbauer F (2013) Circulating microRNAs in hematological diseases: principles, challenges and perspectives. Blood 121(25):4977–4984CrossRefPubMedGoogle Scholar
  12. 12.
    Nolte-'t Hoen ENM, Buermans HPJ, Waasdorp M, Stoorvogel W, Wauben MHM, 't Hoen PAC (2012) Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic Acids Res 40(18):9272–9285CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Skog J, Würdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10(12):1470–1476CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M et al (2013) Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 14:319CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622CrossRefPubMedGoogle Scholar
  16. 16.
    Jensen SG, Lamy P, Rasmussen MH, Ostenfeld MS, Dyrskjøt L, Orntoft TF, Andersen CL (2011) Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs. BMC Genomics 12:435CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lunavat TR, Cheng L, Kim DK, Bhadury J, Jang SC, Lässer C, Sharples RA, López MD, Nilsson J, Gho YS, Hill AF, Lötvall J (2015) Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells--evidence of unique microRNA cargos. RNA Biol 12(8):810–823CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Pritchard CC, Kroh E, Wood B, Arroyo JD, Dougherty KJ, Miyaji MM, Tait JF, Tewari M (2012) Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prev Res (Phila) 5(3):492–497CrossRefGoogle Scholar
  20. 20.
    Fields C, Li P, O'Mahony JJ, Lee GU (2016) Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation. Biotechnol Bioeng 113(1):11–25CrossRefPubMedGoogle Scholar
  21. 21.
    Akers JC, Ramakrishnan V, Yang I, Hua W, Mao Y, Carter BS, Chen CC (2016) Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid. Cancer Biomark 17(2):125–132CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yoshioka Y, Kosaka N, Konishi Y, Ohta H, Okamoto H, Sonoda H, Nonaka R, Yamamoto H, Ishii H, Mori M, Furuta K, Nakajima T, Hayashi H, Sugisaki H, Higashimoto H, Kato T, Takeshita F, Ochiya T (2014) Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. Nat Commun 5:3591CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Exosomics Siena SpASienaItaly

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