Name: Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR (Video) | JoVE Journal
Uploaded: 2014-01-22T13:00:00.000+00:00
Duration: 9 min 26 s
Description: Learn quantitative PCR profiling of microRNAs in cerebrospinal fluid using reverse transcription and real-time amplification. This sensitive method enables analysis of 742 unique sequences for neurobiological research.

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5 minutes

The project described was supported by Award Number R01MH079751 (PI: F. Peruzzi) from the National Institute of Mental Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institute of Health.

The following protocol describes the standard procedure to isolate RNA from CSF and profile microRNAs using ready PCR plates. Note that the organic phase extraction is optional, and that a carrier RNA is added to the sample during extraction ensuring maximal recovery of RNA. Consequently, there is no need to quantify the RNA.
Overall, an average of 6-8 plates can be run in one day if the cDNA is synthesized the day before (about 2 hr). Analysis of data is performed when all samples have been profiled and loaded into the software. Depending on the number of samples/groups or their combination for comparison, data analysis may require from one to several hours.
1. RNA Extraction
The RNA extraction was performed from CSF samples, stored frozen at -80 &#176;C in aliquots until analysis. For this procedure the mirVana Paris isolation kit was used, following the manufacturer&#39;s instructions for the total RNA isolation. Please note that, although enrichment for small RNA species is possible with this kit, this step is not done and the RNA extraction procedure is ended after the total RNA isolation step. In addition, although the mirVana Paris isolation kit (like other commercially available RNA isolation kits) does not require organic extraction, a protocol for this procedure can be found below. 
	The protocol for RNA extraction consists of two steps: 
	1.1. Organic Extraction (not required if using mirVana Paris RNA extraction kit) 
	1.2.2. Total RNA isolation
1.1. Organic extraction
1.1.1. Prepare reagents
<ol>
	<li>Add 375 &mu;l of 2-mercaptoethanol to 2x Denaturing solution, and store it at 4 &#176;C.</li>
</ol>
1.1.2. Prepare the CSF
<ol>
	<li>Thaw every aliquot of CSF on ice before the RNA extraction. Add 1 &#181;g of MS2 RNA carrier to 0.5 ml of thawed CSF and gently mix. Note that if the organic extraction is omitted, the MS2 carrier will be added to the sample prior RNA isolation described in 1.2.2.1 below.</li>
	<li>Mix 0.5 ml of 2x Denaturing Solution at room temperature to the CSF.</li>
	<li>Add 1 ml of acid-phenol:chloroform to the CSF plus the 2x Denaturing Solution: be sure to withdrawn the bottom phase containing acid-phenol:chloroform, not the aqueous buffer that lies on top of the mixture. Additionally, note that acid-phenol:chloroform contains phenol, which is a poison and an irritant, use gloves and other personal protection equipment when working with this reagent.</li>
	<li>Vortex for 30-60 sec to mix. For convenience, the 2 ml of sample/denaturing solution/acid-phenol:chloroform mix are divided into two 1.5 ml Eppendorf tubes.</li>
	<li>Centrifuge for 5 min at maximum speed (10,000 x g) at room temperature.</li>
	<li>Carefully remove the aqueous (upper) phase without disturbing the lower phase or the interphase and transfer it to a fresh tube. Note the volume recovered.</li>
</ol>
1.2. RNA isolation
It is recommended to have a dedicated bench, set of pipettes, and racks for handling RNA samples. The working area and tools are decontaminated by using RNase Zap spray and wipes prior each experiment.
1.2.1. Prepare reagents:
<ol>
	<li>Add 21 ml of 100% ethanol to miRNA Wash Solution 1 and 40 ml of 100 % ethanol to Wash Solution 2/3. Please note that miRNA Wash Solution 1 contains guanidium thiocyanate that is a potentially hazardous substance.</li>
</ol>
1.2.2. Total RNA isolation
<ol>
	<li>Add 1.25 volumes of room temperature 100% ethanol to the aqueous phase from the organic extraction and mix thoroughly.</li>
	<li>Place a Filter Cartridge into one of the Collection Tubes.</li>
	<li>Pipette not more than 700 &mu;l of the lysate/ethanol mixture onto the Filter Cartridge.</li>
	<li>Centrifuge for 30 sec at maximum speed. Apply the mixture exceeding 700 &mu;l in successive applications to the same filter. Discard the flow-through after every centrifugation and save the Collection Tube for the washing steps.</li>
	<li>Apply 700 &mu;l miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 15 sec at maximum speed. Discard the flow through from the Collection Tube and replace the Filter Cartridge into the same Collection Tube.</li>
	<li>Apply 500 &mu;l Wash Solution 2/3 and centrifuge for 15 sec at maximum speed. Discard the flow through from the Collection Tube and replace the Filter Cartridge into the same Collection Tube. Apply a second 500 &mu;l Wash Solution 2/3 and centrifuge for 15 sec at maximum speed. Discard the flow through from the Collection Tube and replace the Filter Cartridge into the same Collection Tube. Spin the assembly for 1 min at maximum speed to remove residual fluid from the filter.</li>
	<li>Transfer the Filter Cartridge into a fresh Collection Tube. Apply 100 &mu;l of preheated (to 95 &#176;C) nuclease-free water to the center of the filter, and close the cap. Centrifuge for 30 sec at maximum speed to recover the RNA.</li>
	<li>Collect the eluate containing RNA and store it at -80 &#176;C for subsequent applications.</li>
	<li>Quantify 1 &#181;l of RNA using the NanoDrop 2000c spectrophotometer. Note that RNA quantification can be skipped if an RNA carrier is added to the sample during RNA extraction. In that case 8 &#181;l of RNA are subjected to cDNA synthesis as detailed below.</li>
</ol>
2. miRNA Profile: qRT-PCR Protocol
The protocol for miRNA profiling consists of two steps:
<ol>
	<li>First-strand cDNA synthesis</li>
	<li>Real-time PCR amplification</li>
</ol>
2.1. First-strand cDNA synthesis
2.1.1. Dilute template RNA
<ol>
	<li>Adjust each of the template RNA samples to a concentration of 5 ng/&#181;l using nuclease free water. If the RNA has not been quantified (see step 1.2.2.9), then 8 &#181;l of RNA are utilized for cDNA synthesis</li>
</ol>
2.1.2. Prepare reagents
<ol>
	<li>Gently thaw the 5x Reaction buffer and nuclease free water, and immediately place on ice.</li>
	<li>Mix by vortexing. Resuspend the RNA spike-in by adding 40 &#181;l nuclease free water to the tube and mix by vortexing; leave it on ice for 15-20 min. Immediately before use, remove the enzyme mix from the freezer, mix by flicking the tubes and place on ice. Spin down all reagents.</li>
</ol>
2.1.3. Reverse transcription reaction setup
<ol>
	<li>Prepare the required amount of RT working solution in a 1.5 ml Eppendorf tube, or equivalent (in the proportions indicated in Table 1, except for the RNA template), mix by vortexing (1-2 sec at max speed), briefly spin down (we use a mini Eppendorf centrifuge with fixed speed, 10 sec) and place it on ice. Note that the UniSp6 RNA spike-in is added to the sample prior the RT reaction (see Table 1).</li>
	<li>Dispense RT working solution into nuclease free PCR tubes.</li>
	<li>Dispense template RNA in each tube. 
		Note: remember to calculate 10% excess volume/each reagent for pipetting and/or robotic dead volume.
		<table border="1" cellpadding="0" cellspacing="0">
			<tbody>
				<tr>
					<td>Reagent</td>
					<td>Panel I Volume (&#181;l)</td>
					<td>Panel I + II Volume (&#181;l)</td>
				</tr>
				<tr>
					<td>5x reaction buffer</td>
					<td>4.4</td>
					<td>8.8</td>
				</tr>
				<tr>
					<td>Nuclease free water</td>
					<td>9.9</td>
					<td>19.8</td>
				</tr>
				<tr>
					<td>Enzyme mix</td>
					<td>2.2</td>
					<td>4.4</td>
				</tr>
				<tr>
					<td>UniSp6 RNA Spike-in template</td>
					<td>1.1</td>
					<td>2.2</td>
				</tr>
				<tr>
					<td>Template total RNA (5 ng/&#181;l)</td>
					<td>4.4</td>
					<td>8.8</td>
				</tr>
				<tr>
					<td>Total Volume</td>
					<td>22</td>
					<td>44</td>
				</tr>
			</tbody>
		</table>
		 
		Table 1. Reverse transcription reaction setup.</li>
</ol>
2.1.4. Mix and spin reagents
<ol>
	<li>Mix the reaction by gentle (1-2 sec) vortexing to ensure that all reagents are thoroughly mixed. After mixing, spin down (mini Eppendorf centrifuge with fixed speed, 10 sec).</li>
</ol>
2.1.5. Incubate and heat inactivate
<ol>
	<li>Program a PCR machine as follow:</li>
	<li>Incubate for 60 min at 42 &#176;C</li>
	<li>Heat-inactivate the reverse transcriptase for 5 min at 95 &#176;C</li>
	<li>Immediately cool to 4 &#176;C</li>
	<li>Store at 4 &#176;C or freeze 
		Note: the protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20 &#176;C for up to 5 weeks (optional store at 4 &#176;C for up to 4 days).</li>
</ol>
2.2. Real-time PCR amplification
2.2.1. Prepare reagents for Real-time PCR
<ol>
	<li>Place cDNA (from step 2.1.5) on ice.</li>
	<li>Thaw nuclease free water and PCR Master mix on ice. Protect the PCR Master mix vials from light by covering them with aluminum foil. Immediately before use, mix the PCR Master mix by vortexing 1-2 sec in a mini centrifuge and spin down at 1,500 x g for 1 min.</li>
</ol>
2.2.2. Combine SYBR Green master mix, water, and cDNA and add to PCR plates
The following procedure is recommended to avoid low concentrations of cDNA from adhering to tube surface:
<ol>
	<li>Before removing the plate seal, briefly spin down the plate(s) in a refrigerated centrifuge at 1,500 x g for 1 min.</li>
	<li>In a 15 ml conical tube, combine 2x PCR Master mix and water. Panel I: 2,000 &#181;l 2x master mix and 1,980 &#181;l water; Panel I+II: 4,000 &#181;l 2x master mix and 3,960 &#181;l water.</li>
	<li>Add 20 &#181;l cDNA (panel I) or 40 &#181;l cDNA (panel I+II) and mix.</li>
	<li>Mix gently by vortexing 1-2 sec and spin down in a refrigerated centrifuge at 1,500 x g for 1 min.</li>
	<li>Place the PCR Master mix/cDNA mix in a multichannel pipette reservoir. You may have to identify a reservoir that has the length of the multichannel pipette. This keeps the level of volume high enough allowing for equal volume aliquots throughout the multichannel. This is critical towards the last few aliquots.</li>
	<li>Dispense 10 &#181;l PCR Master mix/cDNA mix to each well of the 384-well plate using a 16-channel pipette.</li>
	<li>Seal the plate with optical sealing.</li>
	<li>Spin plate briefly in a refrigerated centrifuge (1,500 x g for 1 min), to mix the solutions and remove air bubbles. 
		 
		Note: the experiment can be paused at this point. Store the reactions protected from light at 4 &#176;C for up to 24 hr.</li>
</ol>
2.2.3. Real-Time PCR
Perform real-time PCR amplification and melting curve analysis following the parameters detailed in Table 2.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Process Step</td>
			<td>Settings, Roche LC480</td>
		</tr>
		<tr>
			<td>Polymerase Activation/Denaturation</td>
			<td>95 &#176;C, 10 min</td>
		</tr>
		<tr>
			<td>Amplification</td>
			<td>45 Amplification cycles at 
				95 &#176;C, 10 sec 
				60 &#176;C, 1 min 
				Ramp-rate 
				1.6 &#176;C/sec 
				Optical read</td>
		</tr>
		<tr>
			<td>Melting curve analysis</td>
			<td>Yes</td>
		</tr>
	</tbody>
</table>
Table 2. Real-time PCR cycle conditions using the Roche LightCycler 480.
2.3. Real-time PCR data analysis
Real time PCR data analysis is done with the Exiqon GenEx Professional 5.0, following the recommended instructions. The GenEx step-by-step manual can be downloaded at <a href="http://www.exiqon.com/ls/Documents/Scientific/Exiqon-data-analysis-guide.pdf">http://www.exiqon.com/ls/Documents/Scientific/Exiqon-data-analysis-guide.pdf</a> 
	Data analysis consists of three steps:
<ol>
	<li>Import of data</li>
	<li>Preprocessing of data</li>
	<li>Statistical analysis</li>
</ol>
2.3.1. Import of data
<ol>
	<li>In the Roche LightCycler 480 select the 2nd derivative analysis method and calculate the Cq values. Data are exported as a table and saved as text files.</li>
	<li>To import the data, open GenEx and click on the Exiqon import wizard button and click "Start". Follow the instructions to select format, instrument and plate layout file(s). Note that plate layout files (excel files) are downloaded from the Exiqon web site (<a href="http://www.exiqon.com/plate-layout-files">http://www.exiqon.com/plate-layout-files</a>).</li>
	<li>Import Panels I and II and click "Next".</li>
	<li>The table generated after file import contains predefined columns. Samples names can be edited and classification columns can be added or removed at this step. Click "Next" when you are done.</li>
	<li>Save data and load to Data Editor.</li>
</ol>
2.3.2. Preprocessing of data
<ol>
	<li>As recommended by Exiqon and GenEx, when comparing multiple plates the first thing to do is to calibrate the data between the plates by choosing inter-plate calibration from the preprocessing menu.</li>
	<li>It is recommended to run miRNA arrays in triplicate runs. If a miRNA is not present in at least two out of three replicas it will be set as nonexpressed.</li>
	<li>In the next step in the preprocessing, set a cut off. Defining a cut off value indicates that data with a threshold cycle (Ct) higher than this value are discarded. In the present experiments using cerebrospinal fluid specimen, the cut off was set at 39. Therefore, all samples with a Ct higher than 39 in the three replicas will be discarded. If a miRNA has a Ct &#62; 39 for one probe (one replica out of three), that reading will be replaced with the average of the Ct for the other two probes, provided they are both below 39.</li>
	<li>Define reference genes. GenEx Professional has the option to utilize geNorm and/or NormFinder to identify reference genes. Select a list of miRNAs that have similar Cq values across all sample and run geNorm and/or NormFinder. According to this analysis, in the example provided here, miR-622 and miR-1266 had the most uniform values across the samples and were chosen as reference genes (Figure 1).</li>
	<li>Next, data are normalized using the reference genes and the values obtained correspond to the &#916;Ct</li>
	<li>If cDNA synthesis reactions were performed in triplicates, at this point the values should be averaged.</li>
	<li>Validate sheet to remove empty and almost empty columns. In the preprocessing window select "Validate sheet" and than apply on the Remove empty columns line. In the line below, chose the desired percentage of valid data and click apply. For instance, select 100% if you wish to compare only miRNAs common to all samples.</li>
	<li>Handle missing data. 
		Select &#8220;Missing data&#8221; from the Preprocessing menu and chose one of the options to handle missing data. This step is required. Note that GenEx will give you error if you try to load files that contain unhandled missing data.</li>
	<li>Determine relative quantification between groups of samples (i.e. experiment versus control). Relative quantification among groups is calculated using the &#916;&#916; Ct method. In GenEx, click the preprocessing tab and select relative quantification. In the window select the reference group and hit apply. Note that for graphical representations and/or for further statistical analyses, expression data should be converted to a logarithmic scale. In the preprocessing menu, select Log2.</li>
</ol>
2.3.3. Statistical Analysis
GenEx software allows a wide range of statistical analyses, including T-test and ANOVA.
<ol>
	<li>Load the final mdf file into GenEx and open data manager. It is critical to select the correct samples or groups of samples for which statistical analysis is performed.</li>
	<li>Select "Statistics" and chose the icon corresponding to the desired test.</li>
	<li>Click "Run" in the control panel window. Save results as excel or mdf files.</li>
</ol>
2.3.4. Expression profiling
MicroRNAs and samples can be classified, clustered, and visualized on heat-maps and dendrograms, as follows
<ol>
	<li>Load the final mdf file into GenEx and open data manager. Use this window to select and create groups of samples or miRNAs. Click "Apply" when you are done.</li>
	<li>In the upper left corner select cluster and click on the heatmap icon</li>
	<li>In the control panel window click "Run". The heatmap can be saved in various formats such as tiff or bmp; it can also be copied and modified as needed.</li>
</ol>

<ol><li>Pacifici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebrospinal+fluid+miRNA+profile+in+HIV-encephalitis%5BTitle%5D)+AND+%22J.+Cell.+Physiol%22%5BJournal%5D)">Cerebrospinal fluid miRNA profile in HIV-encephalitis.</a> J. Cell. Physiol. 10, (2012).</li>
<li>Krol, J., Loedige, I., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+widespread+regulation+of+microRNA+biogenesis%2C+function+and+decay%5BTitle%5D)+AND+%22Nat.+Rev.+Genet.%22%5BJournal%5D)">The widespread regulation of microRNA biogenesis, function and decay.</a> Nat. Rev. Genet.. 11, 597-610 (2010).</li>
<li>Calin, G. A., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+signatures+in+human+cancers%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">MicroRNA signatures in human cancers.</a> Nat. Rev. Cancer. 6, 857-866 (2006).</li>
<li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+microRNAs+in+serum%3A+a+novel+class+of+biomarkers+for+diagnosis+of+cancer+and+other+diseases%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.</a> Cell Res. 18, 997-1006 (2008).</li>
<li>De Smaele, E., Ferretti, E., Gulino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNAs+as+biomarkers+for+CNS+cancer+and+other+disorders%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">MicroRNAs as biomarkers for CNS cancer and other disorders.</a> Brain Res. 1338, 100-111 (2010).</li>
<li>Heneghan, H. M., Miller, N., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MiRNAs+as+biomarkers+and+therapeutic+targets+in+cancer%5BTitle%5D)+AND+%22Curr.+Opin.+Pharmacol%22%5BJournal%5D)">MiRNAs as biomarkers and therapeutic targets in cancer.</a> Curr. Opin. Pharmacol. 10, 543-550 (2010).</li>
<li>Kosaka, N., Iguchi, H., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+microRNA+in+body+fluid%3A+a+new+potential+biomarker+for+cancer+diagnosis+and+prognosis%5BTitle%5D)+AND+%22Cancer+Sci%22%5BJournal%5D)">Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis.</a> Cancer Sci. 101, 2087-2092 (2010).</li>
<li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+expression+profiles+classify+human+cancers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">MicroRNA expression profiles classify human cancers.</a> Nature. 435, 834-838 (2005).</li>
<li>Shah, A. A., Leidinger, P., Blin, N., Meese, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miRNA%3A+small+molecules+as+potential+novel+biomarkers+in+cancer%5BTitle%5D)+AND+%22Curr.+Med.+Chem%22%5BJournal%5D)">miRNA: small molecules as potential novel biomarkers in cancer.</a> Curr. Med. Chem. 17, 4427-4432 (2010).</li>
<li>Blondal, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+sample+and+miRNA+profile+quality+in+serum+and+plasma+or+other+biofluids%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Assessing sample and miRNA profile quality in serum and plasma or other biofluids.</a> Methods. 59, 1-6 (2013).</li>
<li>Jensen, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+two+commercial+global+miRNA+expression+profiling+platforms+for+detection+of+less+abundant+miRNAs%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs.</a> BMC Genomics. 12, 435 (2011).</li>
</ol>

The authors have nothing to disclose.

MicroRNAs are small noncoding RNAs that regulate gene expression by inhibiting translation and/or promoting mRNA degradation2. Due to their high stability in cell-free conditions, microRNAs have been detected in many body fluids. Furthermore, distinct expression profile of microRNAs has been correlated with stage and/or progression in a variety of human cancers3-9.
There are several tools available to profile microRNAs, including classical chip arrays or the latest deep s...

MicroRNAs belong to the family of small (21-23 nt in length) noncoding RNAs that regulate gene expression post-transcriptionally. microRNAs can be secreted in the extracellular space where they appear to be relatively stable. While determining changes in miRNA expression can be an important step toward identification of biomarkers, performing miRNA profiles and handling a large amount of data may be intimidating.
Here, we describe a protocol to determine changes in miRNA expression in the cerebrospinal fluid (applicable to other body fluids) by a sensitive real time PCR. We also describe the use of software for data analysis, including statistical analysis and graphical representation of results. The entire procedure is relatively easy and straightforward and, depending on the number of samples to be profiled and the number of real time PCR machines available, also relatively quick. The experimental part requires accuracy in handling RNA and pipetting into 384-well plates, while the data analysis section using GenEx requires some basic knowledge in informatics and statistics.

<table><tbody><tr><td colspan="4">&lt;p&gt;&lt;strong&gt;Table 3. List of equipment.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Finnpipette Novus Multichannel Pipetter&lt;/p&gt; </td><td>Thermo Scientific</td><td>&lt;p&gt;HH05279&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Finntip Pipette Tips&lt;/p&gt; </td><td>Thermo Scientific</td><td>&lt;p&gt;9400613&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Thermal Cycler C1000&lt;/p&gt; </td><td>Biorad</td><td></td><td></td></tr><tr><td> &lt;p&gt;0.2 ml Low Profile, Clear PCR tubes&lt;/p&gt; </td><td>Biorad</td><td>&lt;p&gt;TLS0801&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Flat Cap Strips&lt;/p&gt; </td><td>Biorad</td><td>&lt;p&gt;TLS0803&lt;/p&gt;</td><td></td></tr><tr><td>&lt;p&gt;LightCycler 480 Real-Time PCR System&lt;/p&gt;</td><td>Roche</td><td></td><td></td></tr><tr><td>&lt;p&gt;LightCycler 480 Sealing Foil&lt;/p&gt;</td><td>Roche</td><td>&lt;p&gt;04 729 757 001&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Refrigerated Centrifuge 5804R&lt;/p&gt; </td><td>Eppendorf</td><td></td><td></td></tr><tr><td> &lt;p&gt;Swing-bucket Rotor, 4-place&lt;/p&gt; </td><td>Eppendorf</td><td>&lt;p&gt;A-4-44&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Bench-Top Mini Centrifuge&lt;/p&gt; </td><td>Fisher</td><td>&lt;p&gt;05-090-100&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Bench-Top Vortex&lt;/p&gt; </td><td>Fisher</td><td></td><td></td></tr><tr><td> &lt;p&gt;1.5 ml Microcentrifuge Tubes, Sterilized&lt;/p&gt; </td><td>Fisher</td><td>&lt;p&gt;02-681-5&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Matrix Reagent Reservoir, 25 ml&lt;/p&gt; </td><td>Thermo Scientific</td><td>&lt;p&gt;8093&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Thermomixer Confort&lt;/p&gt; </td><td>Eppendorf</td><td></td><td></td></tr><tr><td>&lt;p&gt;Bench-Top Mini Centrifuge Sigma 1-15&lt;/p&gt;</td><td>Sigma</td><td></td><td></td></tr><tr><td> &lt;p&gt;Bench-Top Vortex&lt;/p&gt; </td><td>Velp Scientifica</td><td>&lt;p&gt;F202A0173&lt;/p&gt;</td><td></td></tr><tr><td colspan="4">&lt;p&gt;&lt;strong&gt;Table 4. List of reagents.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td> &lt;p&gt;Universal c-DNA synthesis kit, 16-32 rxns&lt;/p&gt; </td><td>Exiqon</td><td>&lt;p&gt;203300&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;SYBR Green master mix, Universal RT, 25 ml&lt;/p&gt; </td><td>Exiqon</td><td>&lt;p&gt;203400&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Ultrapure Distilled Water Dnase, Rnase free&lt;/p&gt; </td><td>Invitrogen</td><td>&lt;p&gt;10977-015&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;MicroRNA Ready to use PCR Human Panels (I+II) V2.R&lt;/p&gt; </td><td>Exiqon</td><td>&lt;p&gt;203608&lt;/p&gt;</td><td></td></tr><tr><td>&lt;p&gt;mirVana miRNA isolation Kit, 40 isolations&lt;/p&gt;</td><td>Ambion</td><td>&lt;p&gt;AM1556&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;MS2 RNA carrier&lt;/p&gt; </td><td>Roche Applied Science</td><td>&lt;p&gt;10165948001&lt;/p&gt;</td><td></td></tr><tr><td>&lt;p&gt;2-mercaptoethanol 98%&lt;/p&gt;</td><td>Sigma Aldrich</td><td>&lt;p&gt;M3148-25ml&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Ethanol 100%&lt;/p&gt; </td><td>Carlo Erba</td><td>&lt;p&gt;64-17-5&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Nuclease free water&lt;/p&gt; </td><td>Qiagen</td><td>&lt;p&gt;1039480&lt;/p&gt;</td><td></td></tr><tr><td>&lt;p&gt;RNAse Zap&lt;/p&gt;</td><td>Ambion</td><td>&lt;p&gt;AM9780&lt;/p&gt;</td><td></td></tr></tbody></table>

cerebrospinal fluid microrna profiling using quantitative real time pcr

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

Results from this study have been previously published1. Figure 1 shows results from the analysis of candidate reference microRNAs using geNorm application. Accordingly, two microRNAs, miR-622 and miR-1266, were identified as reference genes and were used to normalize miRNA values.
For the CSF study we had three groups of samples: HIV- (n=10), HIVE (n=4), and HIV+ without Encephalitis (HIV+, n=5). The two groups, HIVE and HIV+, were compar...

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

The overall goal of this procedure is to profile microRNAs in cerebral spinal fluids by quantitative PCR and to analyze the data using Gen X professional software. This is accomplished by first isolating total RNA from cerebral spinal fluid samples. The second step is to perform reverse transcription in order to transcribe the RNAs into CDNAs.Next, the CDNAs are mixed with a cyber green master mix loaded into 384 well plates containing primers for 742 unique sequences and amplified using real-time PCR. The final step is to analyze the data acquired from the real-time amplification. Ultimately, the results can be expressed as a fold change, increase or decrease over a control, and visualized in various graphic representations.So the main advantage of our technique over existing methods like chip arrays is that our method is highly sensitive and requires minimal amount of RNAs. Generally, individuals new to this method will struggle because of the high amount of data generated and the use of the software for data analysis. First, spray the working area and pipettes with RNA Zap.To begin, CDNA synthesis dilute the template RNA samples to a concentration of five nanograms per microliter with nuclease free water. Next, gently place the five x reaction buffer on ice to thaw. Then add 40 microliters of nuclease free water to the RNA spike in to resuspend the reagent, vortex the tube and leave it on ice for 15 to 20 minutes.Next, remove the enzymes from the freezer, flick the tubes and place them on ice. Also, once all the reagents have thawed, quickly spin them down. Prepare reverse transcriptase working solution in a 1.5 milliliter eph tube by adding the five x reaction buffer enzyme.Mix nuclease free water and RNA spike in template using the proportions indicated in table one. Next, mix the working solution by vortexing it for one to two seconds at maximum speed. Briefly spin down the solution for 10 seconds, and then dispense the working solution into nuclease free PCR tubes.Then add the template RNA directly into the PCR tubes. Gently vortex the reaction for one to two seconds to ensure that all of the reagents are thoroughly mixed and spin down the tubes for 10 seconds. Place the sample in the PCR machine and enter the settings shown here.Then start the program following retro transcription. The CDNA is ready for real-time PCR amplification and can be stored at negative 20 degrees Celsius for up to five weeks. To begin real-time PCR amplification, first place the CDNA and the PCR master mix on ice.To thaw protect the PCR master mix vials from light by covering them with aluminum foil while they thaw. Once thawed vortex, the PCR master mix for one to two seconds. Next, combine 2000 microliters of two XPCR master mix and 1, 980 microliters of water in a 15 milliliter conical tube.Add 20 microliters of the CDNA to the mixture and gently vortex the solutions for one to two seconds. Then spin the solutions down in a refrigerated centrifuge at 1, 500 times G for one minute at four degrees Celsius. Next place the PCR reaction mix into a multi-channel pipette reservoir.Briefly spin down the ready-to-use plates in a refrigerated centrifuge at 1, 500 times G before removing the plate seal. Then dispense 10 microliters of the PCR master mix containing CD NA to each well of a 384 well plate. Using a 16 channel pipette and seal the plate with an optical sealing film.After adding the master mix, briefly spin the plate in a refrigerated centrifuge at 1, 500 times G to pull the solution to the bottom of the wells. Mix the samples and remove air bubbles to pause the experiment. At this point, store the reactions in an area protected from light at four degrees Celsius for up to 24 hours.Next, perform real-time PCR amplification and melting curve analysis following the parameters detailed in table two. To begin analysis, select the second derivative analysis method in the Roche light cycler. Then calculate the quantification cycle or CQ values.Export the data as a table and save it as a text file. Next, import the data into Gen X by first clicking on the gon import wizard button and then clicking start at the next window, select the format instrument and plate layout files. Plate layout Excel files can be downloaded from the GON website.Then import panel one and click next. The table generated after the file import contains predefined columns, edit sample names, and add or remove classification columns at this step. Click next when you're done and save the data.Then load it to the data editor First, calibrate the data between the plates by choosing INTERPLATE calibration from the pre-processing menu to ensure all of the plates are calculated using the same parameters. Next, define a cutoff value that will discard data higher than the threshold cycle or ct. If a micro RNA has a CT that is greater for one replica out of three, that reading will be replaced with the average of the CT from the two other probes.Define reference genes by selecting a list of microRNAs that have similar CQ values across all samples and run gene norm. Next, normalize the data using the obtained reference genes. The resulting values will correspond to the delta ct.Remove empty and almost empty columns by opening the pre-processing window and selecting validate sheet, and then selecting Apply on the remove empty columns line in the line below. Choose the desired percentage of valid data and click apply. Select 100%if you wish to compare only microRNAs common to all samples.Load the final MDF file into Gen X and open the data manager to classify cluster and visualize microRNAs and samples on heat maps and dendro grams. Then select and create groups of samples and click apply when you're done. Next in the control panel window, click run.Save the heat map in the desired format such as TIFF or bitmap, and then copy and modify the heat map as needed. This graphic bar was obtained from the G norm application within Gen X to analyze 10 possible micro RNA reference genes. These results indicate that MIR 622 and MI 1, 266 are the most stably expressed microRNAs.The box plot displayed here shows the distribution of data for each of 11 statistically significant microRNAs within the groups of HIVE and HIV plus without encephalitis. In each column, the plot indicates the median and first and third quartiles, along with the maximum and minimum values. Following this procedure, mineral profiling can be performed on any body fluids, tissue samples, and cultured cells.

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