Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle Capture from Plasma Samples
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 wordsExtracellular vesicles miRNA Antibody-coated beads Plasma RT-qPCR
- 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
- 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.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.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
- 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
- 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
- 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