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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Medicine

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Published: June 13th, 2016

DOI:

10.3791/54036

1Division of Gastroenterology and Hepatology, Department of Medicine, Johns Hopkins University School of Medicine, 2Department of Surgery, Tohoku University

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.

Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 °C.

Like other biological fluids, i.e., breast milk, plasma or urine, bile contains exosomes, lipid rich vesicles1-4. Exosomes can induce or alter biological functions in recipient cells5, 6, a form of cell-cell communication that might be part of their normal function4, 7-9. Exosomes can contain miRNA species which can provide a valuable source of biomarkers for diagnosis10. miRNAs profiles have gained substantial attention in recent years as diagnostic and prognostic markers for a variety of diseases11-14.

miRNA itself can be found in biological fluids, possibly released by dead ....

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Obtaining the bile from patients by endoscopic retrograde cholangiopancreatography (ERCP) requires the approval of a human subjects study protocol by the Institutional Review Board. All work presented here was approved by the Johns Hopkins University Institutional Review Board.

1. Exosome Isolation from Bile

  1. Perform endoscopic retrograde cholangiopancreatography (ERCP). Place the patient in the supine position and intubate.
    1. Pass a duodenoscope through the mouth of the p.......

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Since exosomes are too small to be detected by regular microscopy or flow cytometry, electron microscopy or nanoparticle optical analysis has to be performed. The nanoparticle optical analysis has the advantage over electron microscopy that it is also quantitative and provides size distribution and concentration. The instrument introduces a finely focused laser beam through a glass prism into the sample. The Brownian motion of the isolated vesicles is captured via an EMCCD high sensitivit.......

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To reliably utilize exosomes isolated from bile, it is important to employ consistent high quality isolation methods to obtain high quality samples in return. The methodology defined in this paper is a well-established way to isolate exosomes and miRNA from human bile. It highlights several crucial steps in the characterization of the isolated exosomes which at a minimum should comprise electron microscopy or nanoparticle optical analysis and Western blots.

The most crucial step in the isolati.......

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This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

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Name Company Catalog Number Comments
0.2µm Polyethersulfone (PES) membrane filter Corning 431229
thinwall polyallomer ultracentrifuge tubes  Beckman Coulter 331374
SW40Ti rotor Beckman Coulter
LM10-HS  Nanosight
Nanoparticle Tracking Analysis (NTA) software  Nanosight
mirVANA Life Technologies AM1560
cOmplete Protease Inhibitor Roche distributed by Sigma-Aldrich 4693116001
Immobilon PSQ Millipore, Bedford MA ISEQ00010
Anti-CD63 antibody Abcam ab59479
Anti-Tsg101 Abcam ab30871

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