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

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
<ol>
	<li>Perform endoscopic retrograde cholangiopancreatography (ERCP). Place the patient in the supine position and intubate.
	<ol>
		<li>Pass a duodenoscope through the mouth of the patient and insert it into the second part of the duodenum and identify the major papilla.</li>
		<li>Selectively cannulate the common bile duct by inserting a triple lumen sphincterotome pre-loaded with a 0.035 inch (0.9 mm) hydrophilic guidewire.</li>
		<li>Advance the guidewire under fluoroscopic guidance to the left hepatic duct since on occasion it can be difficult to differentiate the cystic duct from the right hepatic duct, then advance the sphincterotome 5 cm into the bile duct to acquire a stable position.</li>
		<li>Remove the guidewire, attach a 10 ml syringe to the guidewire port through the Luer lock and aspirate the bile using 10 ml of negative pressure.</li>
		<li>Remove the syringe containing the bile to proceed with the ERCP by reinserting the guidewire and injecting contrast agent through the injection port to opacify the biliary tree.</li>
		<li>Transfer the collected bile into a 15 ml tube on ice and keep on ice until ready to proceed with the centrifugation step. 
		Caution: Wear gloves to protect yourself and the samples! Treat the bile as a potential source of infection as it can contain Hepatitis A viral particles!</li>
		<li>Keep the sample(s) on ice. Either proceed with step 1.2 or centrifuge at 500 x g at 4 &#176;C for 10 min and then freeze at -80 &#176;C until further use. 
		Note: Samples stored this way for several years have routinely been used with good results19.</li>
	</ol>
	</li>
	<li>Transfer 400 &#181;l of bile into a microcentrifuge tube and collect cells and cell debris by centrifugation at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant by pipetting, transfer to a fresh microcentrifuge tube and centrifuge the sample(s) at 16,500 x g for another 20 min at 4 &#176;C to clear the supernatant of further debris. Discard the tubes in the biohazard waste. Alternatively, spin the samples in an ultracentrifuge instead.</li>
	<li>Collect the supernatant and filter it in a biosafety cabinet with a syringe through a 0.2 &#181;m polyethersulfone (PES) membrane filter which has good flow rates and low protein binding to remove any particles larger than 200 nm left.</li>
	<li>Pipet the filtered supernatant to ultracentrifuge tubes and add PBS so that the tubes are more than 2/3 full. If the tubes contain less liquid than that, the tubes will collapse during centrifugation! Tubes can be washed, autoclaved and reused several times.</li>
	<li>Pellet the exosomes through ultracentrifugation at 120,000 x g for 70 min at 4 &#176;C. After the centrifugation look at the bottom of the ultracentrifuge tube to sometimes see small yellow pellets; these are the exosomes.</li>
	<li>Discard the supernatant by carefully decanting it and disinfect the waste by adding bleach to a final concentration of 10% v/v and let it stand for 20 min.</li>
	<li>For downstream experiments, either process the pellet(s) in a small volume of the appropriate solution (i.e., radioimmunoprecipitation assay (RIPA) buffer for protein isolation, RNA lysis buffer for miR isolation) or resuspend it in 50-150 &#181;l of phosphate buffered saline (PBS) which can be either stored at -80 &#176;C for future use (quantitative real-time polymerase chain reaction (qRT-PCR) or protein extraction etc.) or used for characterization of the exosome preparation by TEM or nanoparticle optical analysis.</li>
</ol>
2. Exosome Identification by Transmission Electron Microscopy (TEM) or CryoEM
<ol>
	<li>Resuspend extracellular vesicles in 100 &#181;l of PBS by pipetting. Ideally, use exosomes that have not been stored at -20 &#176;C or -80 &#176;C, especially when performing the isolation for the first time.</li>
	<li>Adsorb a 20 &#181;l aliquot (about 2 &#181;g) to 400 mesh carbon-coated Parlodion copper grids for 2 min and allow to dry at RT.</li>
	<li>Fix the exosome in 1% (v/v) glutaraldehyde (EM-grade) for 5 min at RT by placing drops carefully on the dried preparation.</li>
	<li>Wash the grids twice with water for 5 min and then contrast stain EVs with 1% phosphotungstic acid (PTA) for 30 sec.</li>
	<li>Acquire images with an electron microscope with an acceleration voltage of 80 kilovolts starting at magnifications of 20,000X and increasing to 100,000X when determining the size of the particles. 
	Note: Other preps like layering onto Formvar carbon-coated 200 mesh copper or nickel grids and contrast staining like 1% or 2% uranyl acetate for 10-15 min are also possible.</li>
	<li>For CryoEM, mix a 20 &#181;l aliquot (about 2 &#181;g) with 60 &#181;l Protein G gold 10 nm (1:3 ratio). This will help to determine the exosome diameter.</li>
	<li>Transfer the samples onto glow-discharged C-flat holey carbon grids and remove excess liquid by blotting. Freeze the grids by dipping them immediately into liquid ethane.</li>
	<li>Mount the grids in a cryoholder in liquid nitrogen.</li>
	<li>Acquire the images at 200 kV with magnifications of 20,000X and 100,000X.</li>
</ol>
3. Exosome Identification by Multi-parameter Nanoparticle Optical Analysis
<ol>
	<li>Resuspend the isolated exosomes in 100 &#181;l of PBS by pipetting.</li>
	<li>Dilute exosomes in 600 &#181;l PBS at several different ratios (like 1:50, 1:100, 1:300, 1:600 and 1:1,200).</li>
	<li>Visualize the exosomes by laser light scattering with the appropriate instrument and software in real-time according to manufacturer&#39;s protocol20.</li>
	<li>Increase the level of the camera in capture mode until particles become visible, then adjust the focus to make the particles look smooth.</li>
	<li>While in standard mode, decrease the camera to the lowest level that the particles can still be seen, if necessary adjust camera shutter and gain to achieve this.</li>
	<li>Visually check to ensure 20-100 particles are visible on the screen, if not flush the unit and adjust the dilution in PBS.</li>
	<li>Then adjust the capture duration to between 30-60 sec. After the video is captured, adjust the processing parameters until all particles are identified on the screen and process the video file.</li>
</ol>
4. Exosome Characterization by Western Blot
<ol>
	<li>Lyse exosome pellets from 2-5 ml of bile in 100 &#181;l of ice-cold radioimmunoprecipitation assay (RIPA) buffer with a protease inhibitor by pipetting up and down thoroughly and mix lysates vigorously for 30 sec&#160;on a vortex to solubilize the proteins effectively.</li>
	<li>While most of the time not necessary, use pulse-sonication of the samples on ice with 2 pulses for 10 sec each to ensure full lysis.</li>
	<li>Pellet insoluble material by centrifugation at 15,000 x g at 4 &#176;C for 5 min and transfer supernatant to a clean tube.</li>
	<li>Determine the protein concentration with a method of choice (i.e., bicinchoninic acid (BCA), Coomassie, modified Lowry, 660 nm Protein Assay, etc.) according to manufacturer&#39;s protocol. The method is not as important as long as the same method is used consistently to achieve results that can be compared across experiments.</li>
	<li>Load 50 &#181;g of protein per sample in Laemmli buffer after heating the sample at 95 &#176;C for 5 min into a well of a polyacrylamide gel for electrophoresis (PAGE).</li>
	<li>Electrophorese the samples on the PAGE gels for 1.5 hr and transfer the separated proteins on to a nylon membrane.</li>
	<li>Block the membrane(s) with 5% nonfat milk in Tris-buffered Saline with 0.1% Tween 20 (TBS-T) for 1 hr, then incubate with exosome-specific antibodies like anti-CD63 and anti-TSG-101 at a dilution of 1:500 preferably O/N at 4 &#176;C.</li>
	<li>Wash the membranes with TBS-T 3 x 10 min, then incubate with an appropriate, matching HRP-conjugated secondary antibody in 5% nonfat milk in TBS-T for 1-2 hr at RT.</li>
	<li>Wash the membrane(s) 3 x 5 min with TBS-T and detect the specific proteins with chemiluminescence reagents according to manufacturer&#39;s protocol.</li>
	<li>Image the membranes with film or a digital imaging system according to manufacturer&#39;s protocol.</li>
</ol>
5. MicroRNA Isolation from the Isolated Exosomes
Note: Isolation of miRNA from bile exosomes is carried out using a modified miRNA isolation based on a commercially available kit, but any other RNA isolation method can be used or adapted.
<ol>
	<li>Add 300 &#181;l lysis/building buffer to the exosome pellet (isolated from 400 &#181;l bile), vortex, pipet or pass through a needle/syringe to completely lyse the exosomes to obtain a homogenous lysate.</li>
	<li>Add 1/10 (30 &#181;l) volume of miRNA Homogenate Additive, and mix well by vortexing or inverting the tube several time.</li>
	<li>Incubate the mix for 10 min on ice.</li>
	<li>Add a volume of acid-phenol:chloroform equal to the initial homogenate (300 &#181;l).</li>
	<li>Add 5 &#181;l of 5 fmol/&#181;l cel-miR 39 and vortex for 30-60 sec.</li>
	<li>Centrifuge for 5 min at 10,000 x g at RT to separate the aqueous (upper) and organic (lower) phases.</li>
	<li>Carefully aspirate the upper phase by pipetting and transfer to a fresh tube while noting the volume transferred. Discard the organic phase in the appropriate organic waste container.</li>
	<li>To enrich for small RNAs add 1/3 volume of 100% ethanol to the recovered aqueous phase and mix by thoroughly vortexing or inverting the tube several times.</li>
	<li>Pipet the lysate/ethanol mixture onto the filter cartridge, up to 700 &#181;l at a time.</li>
	<li>Centrifuge the cartridges for 15 sec at a maximum of 10,000 x g or apply vacuum to pass the mixture through the filter.</li>
	<li>Collect the filtrate and if the lysate/ethanol mixture exceeds 700 &#181;l, transfer the flow-through to a fresh tube and repeat the procedure to collect all the filtrates. These filter contains RNA fractions devoid of small RNAs and can be used to recover these larger RNAs.</li>
	<li>Add 2/3 volume of RT 100% ethanol to the total filtrate and mix thoroughly by vortexing.</li>
	<li>Pipet the filtrate/ethanol mixture onto a second filter cartridge. Like before the maximum volume that can be applied at once is 700 &#181;l.</li>
	<li>Centrifuge for 15 sec at 10,000 x g or apply vacuum as described in step 5.10.</li>
	<li>Discard the flow-through, and repeat until all of filtrate/ethanol mixture has been filtered.</li>
	<li>Apply 700 &#181;l miRNA Wash Solution 1 to the filter cartridge, centrifuge for 5-10 sec or apply vacuum and discard the flow-through.</li>
	<li>Wash the filter with 500 &#181;l wash solution 2/3 as done in step 5.16.</li>
	<li>Repeat the wash a second time with 500 &#181;l of wash solution 2/3 as in step 5.16, discard the flow-through and place the filter back in the collection tube.</li>
	<li>Spin the filter cartridge for 1 min at 10,000 x g to remove all residual fluid from the filter.</li>
	<li>Transfer the filter cartridge into a fresh collection tube and apply 100 &#181;l of hot (95 &#176;C) nuclease free water to the center of the filter.</li>
	<li>Spin the assemblies for 20-30 sec at maximum speed to recover the RNA.</li>
	<li>Collect the eluate and use immediately or store at -80 &#176;C.</li>
</ol>

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None of the authors have competing financial interests.

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...

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 cells and the amount of shed cells can be greatly influenced by an underlying disease. The miRNA profile of exosomes does not necessarily reflect the miRNA profile of the originating cell6, 10, 15-17, yet exosomes may carry miRNA signatures that might be characteristic for the parental cell like a tumor cell. Tumor-derived exosomes have been identified in the plasma of patients with lung adenocarcinoma, prostate cancer and other tumors8, 16, 18.
The goal of this method is the reliable and robust isolation of exosomes from human bile specimens, largely independent of their prior handling procedures. It was developed to utilize bile as a reliable source of miRNA for the potential diagnosis of diseases of the bile duct such as cholangiocarcinoma19. It consist of several steps of centrifugation with increasing speeds to isolate exosomes and might be applicable to other biological fluids.

<table><tbody><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) membrane filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>thinwall polyallomer ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>331374</td><td></td></tr><tr><td>SW40Ti rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>LM10-HS&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>Nanoparticle Tracking Analysis (NTA) software&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>mirVANA</td><td>Life Technologies</td><td>AM1560</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche distributed by Sigma-Aldrich</td><td>4693116001</td><td></td></tr><tr><td>Immobilon PSQ</td><td>Millipore, Bedford MA</td><td>ISEQ00010</td><td></td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>Anti-Tsg101</td><td>Abcam</td><td>ab30871</td><td></td></tr></tbody></table>

isolation and profiling of microrna-containing exosomes from human bile

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 &#176;C.

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...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

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 ...

isolation-profiling-microrna-containing-exosomes-from-human

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10.3K Views.  Johns Hopkins University School of Medicine. 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.

Video: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Biochemistry

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Cancer Research

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Immunology and Infection

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biology

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...