Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Summary

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.

Abstract

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.

Introduction

Like other biological fluids, i.e., breast milk, plasma or urine, bile contains exosomes, lipid rich vesicles^1-4. Exosomes can induce or alter biological functions in recipient cells^{5, 6}, a form of cell-cell communication that might be part of their normal function^{4, 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 diseases^11-14.

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

Protocol

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 patient and insert it into the second part of the duodenum and identify the major papilla.
   2. Selectively cannulate the common bile duct by ins....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm 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