This study was supported by funds from Rita Allen Foundation grant to Seena Ajit. The authors would like to acknowledge Erika Balogh and Dr. Soumitra Ghoshroy from the University of South Carolina Electron Microscopy Center for instrument use, scientific and technical assistance.

All experiments using blood samples from human and rodents were executed in compliance with all relevant guidelines, regulations and regulatory agencies. Human subjects were enrolled after giving informed consent as approved by the Drexel University College of Medicine Institutional Review Board and all procedures for studies performed using animals were approved by Drexel's Institutional Animal Care and Use Committee.
1. Exosome Purification from Blood (~5.5 hr)
TIPS
<ul>
	<li class="jove_content">We resuspend exosomes from one tube of blood (~10 ml human blood and ~2 ml mouse blood) in 300 &mu;l RNA lysis buffer from the mirVana miRNA isolation kit.</li>
	<li class="jove_content">To purify exosomes from cell culture media, the media is spun at 500 x g for 10 min to remove cells and also filtered through a 0.22 &mu;m filter after centrifugation at 12,000 x g. The exosome purification from cell culture media does not require a dilution in PBS, the PBS wash or the second ultracentrifugation step.</li>
	<li class="jove_content">If the purified exosomes will be used for proteomics studies, adding a sucrose gradient centrifugation step after the ultracentrifugation step will decrease the amount of protein aggregates and contaminants 15.</li>
	<li class="jove_content">The observed morphology of exosomes can differ depending on both the source and the processing steps for TEM. We did not observe the cup shaped morphology for exosomes which has been previously attributed to differences in sample processing 9,15. Our TEM images including the immunogold labeling are similar to previous reports from different sources including blood, biliary and cortical culture derived exosomes 9,16,17.</li>
</ul>
<ol>
	<li>Invert EDTA coated tube containing the blood 5x and place upright at room temperature for 10-60 min.</li>
	<li>Centrifuge at 2,000 x g for 15 min at 4 &deg;C.</li>
	<li>Collect the top layer, the plasma, in 15 ml tubes and keep on ice.</li>
	<li>Dilute fluid with an equal volume of PBS. Centrifuge 30 min at 2,000 x g at 4 &deg;C.</li>
	<li>Transfer to centrifuge tubes, add 1X PBS for a total volume of 24 ml, and centrifuge 45 min at 12,000 x g at 4 &deg;C.</li>
	<li>Transfer to ultracentrifuge tubes and centrifuge 2 hr at 110,000 x g at 4 &deg;C.</li>
	<li>Resuspend pellet in PBS and centrifuge 1 hr at 100,000 x g at 4 &deg;C.</li>
	<li>Resuspend pellet in appropriate buffer (RNA lysis buffer).</li>
	<li>Resuspend in PBS or RIPA buffer for electron microscopy and western blot analysis respectively.</li>
</ol>
2. RNA Purification Using a Modified mirVana miRNA Isolation Kit (~1 hr)
TIPS
<ul>
	<li class="jove_content">Prepare the area for isolating RNA by wearing personal protective equipment (PPE), wiping surfaces and pipettes with RNase Zap, and using sterile filter tips and tubes.</li>
	<li class="jove_content">Though the protocol from the vendor gives the option of purifying only miRNA, we always isolate total RNA including mRNA and miRNA so that we can study both from the same sample.</li>
	<li class="jove_content">Adding an on-column DNase treatment step removes traces of DNA that may be present. Depending on the source of exosomes, DNA has been reported to be absent in exosomes purified from some sources including mouse and human mast cell lines 8 but chromosomal DNA sequences have been detected in exosomes purified from media of cultured cardiomyocytes 18.</li>
	<li class="jove_content">We reduced the volume of wash solution 1 to 350 &mu;l (Step 2.4 below) because the vendor provides only enough Wash solution 1 for one 700 &mu;l addition. If the volume is not reduced there is not sufficient solution left for using the entire kit contents. Remaining steps were performed according to manufacturer's recommendations.</li>
</ul>
<ol>
	<li>Add 1/10 volume of miRNA Homogenate Additive (30 &mu;l) and incubate on ice for 10 min.</li>
	<li>Extract with a volume of acid-phenol:chloroform equal to the initial lysate volume. Centrifuge and recover the upper phase in a fresh tube.</li>
	<li>Add 1.25 volumes 100% ethanol (375 &mu;l). Vortex. Apply to filter cartridge and centrifuge 15 sec at 10,000 x g.</li>
	<li>Add half of recommended volume miRNA Wash Solution 1 (350 &mu;l) and centrifuge 15 sec at 10,000 x g.</li>
	<li>Add 10 &mu;l DNase 1 to 70 &mu;l buffer RDD (Qiagen) for each sample. Add 80 &mu;l to column and incubate 15 min at room temperature.</li>
	<li>Add Wash Solution 1 (350 &mu;l) and centrifuge 15 sec at 10,000 x g.</li>
	<li>Add Wash Solution 2/3 (500 &mu;l) and centrifuge 15 sec at 10,000 x g. REPEAT.</li>
	<li>Elute with 40 &mu;l 95 &deg;C nuclease-free H2O.</li>
	<li>Determine the concentration of RNA and dilute the RNA to 100 ng/ &mu;l.</li>
</ol>
3. miRNA Profiling of Exosomes from Blood Using Taqman Low Density Array (TLDA) Cards (~6.5 hr)
TIPS
<ul>
	<li class="jove_content">Prepare the area for isolating RNA by wearing PPE, wiping surfaces and pipettes with RNase Zap, and using sterile filter tips and tubes.</li>
	<li class="jove_content">Pre-amplification (pre-amp) step is recommended for 1-350 ng RNA (350-1,000 ng samples do not require pre-amp step after cDNA synthesis). It is important to determine whether pre-amp is necessary for your sample because you must treat all samples under equal conditions. Pre-amp step adds cost and the Ct values have a lower acceptable cut off (32 instead of 40).</li>
	<li class="jove_content">It is useful to prepare the cDNA reaction in strip tubes instead of a plate depending on the number of cards that can be run in a single day. This will minimize freeze-thaw of cDNA samples that will not be processed on the same day.</li>
	<li class="jove_content">Exosomes collected from different sources have different amounts of RNA. This protocol allows a maximum of 3 &mu;l RNA template for the cDNA synthesis reaction. Depending on the source, the amount of exosomal RNA that can be isolated maybe limited. While a typical tissue would yield enough RNA that cDNA would be made from 100 ng RNA in 3 &mu;l volume, exosomes from human blood or human cell culture media often contain less RNA and cDNA must be made from less starting material. Mouse blood exosomes contain higher levels of RNA and at least 100 ng in 3 &mu;l can be obtained.</li>
</ul>
<ol>
	<li>To prepare cDNA from purified RNA using Megaplex Pools reverse transcriptase primers (Pool A and Pool B contain primers for the miRNAs on microfluidic cards Array A and B), thaw the reaction components on ice.</li>
	<li>Label two RNase-free 1.5 ml tubes for RT reaction with Pool A primers or Pool B primers and add components in chart order. Add at least one extra volume of components for 2 or more reactions or make a master mix as recommended by the vendor.</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>RT reaction Component</td>
			<td>Component Concentration</td>
			<td>Volume for 1 sample(total V/sample = 4.5 &mu;l)</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>&nbsp;</td>
			<td>0.20 &mu;l</td>
		</tr>
		<tr>
			<td>Megaplex RT buffer (10X)</td>
			<td>1X</td>
			<td>0.80 &mu;l</td>
		</tr>
		<tr>
			<td>dNTPs (100 mM)</td>
			<td>4.4 mM</td>
			<td>0.20 &mu;l</td>
		</tr>
		<tr>
			<td>MgCl2 (25 mM)</td>
			<td>5 mM</td>
			<td>0.90 &mu;l</td>
		</tr>
		<tr>
			<td>RNase Inhibitor (20U/&mu;l)</td>
			<td>2 U</td>
			<td>0.10 &mu;l</td>
		</tr>
		<tr>
			<td>Megaplex RT Primers A or B (10X)</td>
			<td>1X</td>
			<td>0.80 &mu;l</td>
		</tr>
		<tr>
			<td>MultiScribe Reverse Transcriptase</td>
			<td>75 U</td>
			<td>1.50 &mu;l</td>
		</tr>
	</tbody>
</table>
<ol start="3">
	<li>Invert tubes 6 times and centrifuge briefly. Then, pipette 4.5 &mu;l RT reaction mix into MicroAmp 8-tube Strips or a 96-well MicroAmp optical reaction plate.</li>
	<li>Add 100 ng of RNA and adjust the volume to 3 &mu;l with DEPC treated water or add 3 &mu;l total RNA, stir with the pipette tip, and seal the tubes or plate. Spin briefly and incubate on ice for 5 min.</li>
	<li>Load the reaction into the PCR machine and run it under the following conditions (as recommended by the Applied Biosystems Megaplex Pools protocol):</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Stage</td>
			<td>Temp</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>Cycle (40x)</td>
			<td>16 &deg;C</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>42 &deg;C</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>50 &deg;C</td>
			<td>1 sec</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>85 &deg;C</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &deg;C</td>
			<td>&infin;</td>
		</tr>
	</tbody>
</table>
<ol start="6">
	<li>Store cDNA at -20 &deg;C (good for at least one week) or continue to pre-Amp step which can be stored for one week.</li>
	<li>Thaw PreAmp primers for Pool A and Pool B on ice and invert to mix. Swirl Taqman Pre-Amp master mix and keep on ice also.</li>
	<li>Follow the chart adding at least one extra volume of components for 2 or more reactions or make a master mix as recommended by the vendor.</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Pre-Amp Reaction Component</td>
			<td>Volume for 1 sample</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>7.5 &mu;l</td>
		</tr>
		<tr>
			<td>Taqman PreAmp Master Mix (2X)</td>
			<td>12.5 &mu;l</td>
		</tr>
		<tr>
			<td>Megaplex PreAmp Primers A or B (10X)</td>
			<td>2.5 &mu;l</td>
		</tr>
	</tbody>
</table>
<ol start="9">
	<li>Pipette 2.5 &mu;l RT product into MicroAmp 8-tube Strips or a 96-well MicroAmp optical reaction plate and dispense 22.5 &mu;l PreAmp reaction mix into each well.</li>
	<li>Seal the plate, incubate on ice 5 min and then load the sample into the PCR machine. Run under the following conditions.</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Stage</td>
			<td>Temp</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>95 &deg;C</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>55 &deg;C</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>72 &deg;C</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Cycle (12x)</td>
			<td>95 &deg;C</td>
			<td>15 sec</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>60 &deg;C</td>
			<td>4 min</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>99.9 &deg;C</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &deg;C</td>
			<td>&infin;</td>
		</tr>
	</tbody>
</table>
<ol start="11">
	<li>After the preamp reaction is finished, briefly centrifuge the tubes or plate and then add 75 &mu;l 0.1X TE pH 8.0 to each well or tube.</li>
	<li>Seal and mix and spin briefly. Store the diluted product for one week at -20 &deg;C or continue on to load the plate.</li>
	<li>Combine the following in an RNase-free 1.5 ml tube on ice.</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume for 1 card</td>
		</tr>
		<tr>
			<td>Taqman Universal PCR Master Mix, No AmpErase UNG (2X)</td>
			<td>450 &mu;l</td>
		</tr>
		<tr>
			<td>Diluted preAmp product</td>
			<td>9 &mu;l</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>441 &mu;l</td>
		</tr>
	</tbody>
</table>
<ol start="14">
	<li>Invert the tube to mix and centrifuge briefly. Dispense 100 &mu;l PCR reaction mix into each port of the Taqman MicroRNA Array card.</li>
	<li>Centrifuge the card 2 x 1,000 x g for 1 min. Seal the card and run it using the template provided with the primers in the Applied Biosystems manual and the 384 well Taqman Low Density Array settings. We use Applied Biosystems 7900HT Fast Real-Time PCR System.</li>
</ol>
4. mRNA Analysis by Taqman (~1.5 hr)
<ol>
	<li>To prepare qPCR reaction, mix the reaction components in the order in the chart. All samples will be analyzed with primer probes for the gene of interest as well as 18S rRNA as the normalizer gene.</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Taqman Reaction Component</td>
			<td>Volume for 1 sample</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>7 &mu;l (adjust based on individual sample)</td>
		</tr>
		<tr>
			<td>Taqman Fast Master Mix (2X)</td>
			<td>10 &mu;l</td>
		</tr>
		<tr>
			<td>Taqman Primer probes (20x)</td>
			<td>1 &mu;l</td>
		</tr>
		<tr>
			<td>cDNA from 100 ng RNA</td>
			<td>2 &mu;l (adjust based on individual sample)</td>
		</tr>
	</tbody>
</table>
<ol start="2">
	<li>Run the 96 well plate using the Fast 96-well block as recommended by the manufacturer (Applied Biosystems 7900HT Fast Real-Time PCR System).</li>
</ol>

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Authors have nothing to disclose.

In this protocol, we show the quantification of miRNAs and mRNAs from exosomes purified by differential centrifugation from blood and culture media. Exosomes have diverse components dependent upon their origin and are involved in a number of biological functions, including immune response, antigen presentation, intracellular communication, and the transfer of RNA and proteins 9,11,12,19,20. While size and shape is a determinant of exosome purity, a number of papers showing EM data of exosomes indicate that the...

Short noncoding miRNAs modulate gene expression by binding to the target mRNA. Seed sequence complementarity of ~7 base pairs enables miRNA to bind to the target mRNA resulting in the inhibition of translation or in reduction in the stability of the mRNA, both of which can result in decreased expression of the target protein 1. Research over the last decade has unequivocally proven a fundamental role for miRNAs in mediating cellular functions. There has also been considerable effort directed towards dissecting miRNA mediated molecular changes underlying various diseases 2,3. Furthermore, recent identification of stable miRNAs in bodily fluids 4-6 paved the way for their use as novel biomarkers amenable to clinical diagnosis.
One mode of miRNA transport in bodily fluids is via exosomes, small vesicles that carry mRNAs, proteins, lipid mediators, and miRNAs to recipient cells via systemic blood circulation 7-14. This results in modulation of gene expression in recipient cells and represents a novel mechanism of cellular communication. For instance, cells can modulate immune-regulatory processes by secreting and/or absorbing exosomes containing biomolecules involved in inflammation such as interleukin-1&beta; (IL1&beta;), tumor necrosis factor-&alpha; (TNF&alpha;), transforming growth factor-&beta;5 (TGF&beta;5), and the miRNAs that regulate these genes 13. As aberrant miRNA expression is a common feature in a variety of human diseases, these molecules offer exciting new opportunities for the discovery and validation of novel therapeutic targets 2.
Circulating miRNAs are present in all body fluids and it is known that the composition of exosomes is different based on the source cells from which they were released. Thus they offer an avenue to study the physiological status of the cells and how the cells alter signaling events in response to stress including diseases. Studying alterations in exosome composition can provide insight into signal transduction and investigate their potential utility as biomarkers or therapeutic intervention routes.
Here we will demonstrate the purification of exosomes from multiple sources based on published protocols. These exosomes will be used for RNA isolation followed by qPCR to identify and measure the levels of miRNAs present in the exosomes.

<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (10 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>366643</td>
 <td>For exosome purification from human blood</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (2 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>367841</td>
 <td>For exosome purification from mouse blood</td>
 </tr>
 <tr>
 <td>PAXgene Blood RNA Tube</td>
 <td>BD Diagnostics</td>
 <td>762165</td>
 <td>For miRNA isolation from total blood</td>
 </tr>
 <tr>
 <td>miRVana microRNA isolation kit</td>
 <td>Ambion</td>
 <td>AM1561</td>
 <td></td>
 </tr>
 <tr>
 <td>Acid-Phenol: CHCl3</td>
 <td>Ambion</td>
 <td>9721G</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase 1</td>
 <td>Qiagen</td>
 <td>79254</td>
 <td></td>
 </tr>
 <tr>
 <td>TaqMan Universal PCR Master Mix, No AmpErase UNG</td>
 <td>Applied Biosystems</td>
 <td>4326614</td>
 <td>For TLDA cards</td>
 </tr>
 <tr>
 <td>Megaplex RT rodent Pool Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444746</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex preamp rodent Pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444747</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Rodent MicroRNA A+B set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444909</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex RT Human Pool set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444745</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex Preamp human pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444748</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Human MicroRNA A+B Cards Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444913</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman MicroRNA RT kit</td>
 <td>Applied Biosystems</td>
 <td>4366596</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman fast universal PCR master mix </td>
 <td>Applied Biosystems</td>
 <td>4366072</td>
 <td>For mRNA qRT-PCR</td>
 </tr>
 <tr>
 <td>Tumor necrosis factor (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs01113624_g1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vascular endothelial growth factor A (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs00900055_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>Maxima First Strand cDNA Synthesis Kit for RT-qPCR</td>
 <td>Thermo Scientific</td>
 <td>K1642</td>
 <td>For mRNA</td>
 </tr>
 <tr>
 <td>HSP70 antibody</td>
 <td>Abcam</td>
 <td>ab94368 </td>
 <td>For western blot</td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG-Gold</td>
 <td>Sigma</td>
 <td>G7402</td>
 <td>For electron microscopy</td>
 </tr>
 <tr>
 <td>Rabbit-anti CD81</td>
 <td>Sigma</td>
 <td>SAB3500454</td>
 <td></td>
 </tr>
 <tr>
 <td>Nickel 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Ni</td>
 <td></td>
 </tr>
 <tr>
 <td>Copper 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Cu</td>
 <td></td>
 </tr>
 <tr>
<td></td>
<td></td>
<td></td>
<td>Table 1. Table of specific reagents.</td>
</tr>
 </tbody>
 </table>

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

After isolating exosomes from blood or cell culture media, the purity of the exosomes can be tested by electron microscopy (EM) and western blot (Figures 1A and 1B). We confirmed our exosome preparations from various sources with EM and western blot using multiple antibodies. Figure 1A shows EM images confirming that exosomes are intact with a diameter of ~30 -100 nm and contain CD81 by immunogold labeling. Commonly used exosomal markers are Hsp70 and tetraspannin family...

Watch this Scientific Journal Video about Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media at JoVE.com

Purification and microRNA Profiling of Exosomes Derived from Blood and 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  ...

purification-microrna-profiling-exosomes-derived-from-blood-culture

Research

JoVE Journal

Biology

31.7K Aufrufe.  Drexel University College of Medicine. Das Vorhandensein von stabilen microRNAs (miRNAs) in exosomes hat immense Interesse als neue Art der interzellulären Kommunikation erzeugt, für ihre potenziellen Nutzen als Biomarker und als Weg für eine therapeutische Intervention. Hier zeigen wir exosome Reinigung von Blut und Kulturmedien durch quantitative PCR gefolgt, um miRNAs transportiert identifizieren.

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...