Name: Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease (Video) | JoVE Journal
Uploaded: 2018-07-03T14:45:00.000+00:00
Duration: 4 min 41 s
Description: Learn digital PCR for quantifying circulating microRNAs as cardiovascular disease biomarkers using serum samples. This method enables precise miRNA measurement without standard curves for diagnostic research.

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5 minutes

The authors have no acknowledgments.

1. Extraction of miRNA from Plasma/Serum
Note: In order to quantify miRNAs appropriately, the correct microRNA isolation from the plasma/serum is a crucial step. A key thing to keep in mind, especially because different protocols exist, is to adhere to the same workflow while processing the samples. In this protocol, miRNA is extracted from 50 &#181;L of serum. Do not use more than 200 &#181;L as this limit the correct extraction process.
<ol>
	<li>Prepare serum or thaw frozen samples.</li>
	<li>Add 250 &#181;L (5 volumes) commercial lysis reagent to 50 &#181;L of serum. Support the lysis by vortexing. Place the tube with the lysate on the bench at room temperature for 5 min.</li>
	<li>Spike the lysate with 3.5 &#181;L of spike-in control (1.6 x 107 copies/&#181;L) and mix them by vortexing.</li>
	<li>Add 50 &#181;L of chloroform (i.e., an equal amount as to the starting serum sample) for the phase separation. Shake the tube vigorously for 15 s. Place the tube on the bench at room temperature for 2 - 3 min.</li>
	<li>For the phase separation, centrifuge the tube at 12,000 x g at 4 &#176;C for 15 min. 
	Note: The samples are now separated into an upper aqueous phase containing the RNA, a white interphase, and a lower, pink organic phase.</li>
	<li>Transfer the upper aqueous phase (100 &#181;L) containing the RNA into a new tube. Do not transfer any white interphase material as this falsifies the correct RNA count.</li>
	<li>Add 150 &#181;L (i.e., 1.5x the volume of the transferred sample) of 100% ethanol. Mix the materials by pipetting them up and down. Do not centrifuge them, and quickly move on to the next step.</li>
	<li>Pipette 250 &#181;L of the sample onto a commercial spin column in a 2 mL collection tube. Close the lid. Centrifuge the column at &#62;8,000 x g at room temperature for 15 s. Discard the flow-through.</li>
	<li>Add 700 &#181;L of commercial washing buffer number 1 to the spin column. Close the lid and centrifuge the column at &#62;8,000 x g at room temperature for 15 s. Discard the run-through washing buffer.</li>
	<li>Add 500 &#181;L of commercial washing buffer number 2 to the spin column. Close the lid and centrifuge the column at &#62;8, 000 x g at room temperature for 15 s. Discard the run-through washing buffer.</li>
	<li>Pipette 500 &#181;L of 80% ethanol to the spin column. Close the lid, centrifuge the column at &#62;8, 000 x g at room temperature for 2 min. Discard the collection tube with the flow-through.</li>
	<li>Transfer the spin column to a new 2 mL collection tube. Centrifuge the column at full speed with an opened lid to dry the membrane. Discard the collection tube with the flow-through.</li>
	<li>Transfer the spin column into a new 1.5 mL collection tube. Elute the RNA by applying 30 &#181;L of RNase-free water directly to the center of the membrane. Close the lid. Centrifuge the column at full speed for 1 min.</li>
	<li>Store the RNA at -70 &#176;C. 
	Note: The storage of purified RNA in RNase-free water between -70 &#176;C to -20 &#176;C ensures no degradation of the RNA for 1 year. Following the manufacturer&#8217;s protocol, the minimum amount of RNase-free water to dilute the RNA is 10 &#181;L. Using less than 10 &#181;L may insufficiently hydrate the membrane and reduce the total RNA yield.</li>
</ol>
2. Reverse Transcription
Note: Complementary DNA (cDNA) was synthesized using the following 15 &#181;L reverse transcription (RT) protocol (total reaction volume).
<ol>
	<li>Thaw the RT kit on ice. Keep the enzymes in the freezer as long as possible to prevent them from degrading and spin them down before use. Thaw RT primers on ice and spin them down before use.</li>
	<li>Prepare the master mix as described in Table 1.</li>
	<li>Combine 10 &#181;L of master mix and 5 &#181;L of extracted RNA per well in a 96 well-plate.</li>
	<li>Centrifuge the well plate at 2,000 x g at 4 &#176;C for 2 min.</li>
	<li>Use the thermal cycle protocol described in Table 2 for the reverse transcription. 
	Note: As described below, cDNA may directly be processed in the digital PCR machine (see Table of Materials); alternatively, it may be stored at -20 &#176;C.</li>
</ol>
3. Droplet Generation and Digital PCR
Note: Digital PCR was performed using the following 40 &#181;L protocol.
<ol>
	<li>Thaw the commercial dPCR mix, the prepared cDNA, and the PCR primers on ice. Centrifuge them shortly before use.</li>
	<li>Prepare the master mix as described in Table 3.</li>
	<li>Add 1.33 &#956;L/well of the RT product and centrifuge it briefly.</li>
	<li>Pipette 20 &#956;L of the sample into each well of the 8-well cassette (medium row). 
	Note: If the 8-well cassette is not completely filled, fill up the remaining wells with non-template controls (NTCs) that omit cDNA (i.e., the RT product produced with water instead of extracted RNA) or dPCR commercial buffer. The NTC serves as a contamination control. Water must not be used in the remaining wells, because that will impair the quality of the droplet formation.</li>
	<li>Pipette 70 &#956;L of the droplet generation oil for the probes into the oil wells (bottom row) of the 8-well cassette.</li>
	<li>Place a gasket on top of the 8-well cassette and place the cassette in the droplet generator. Close the droplet generator. Wait until the 3 indicator lights are solid green. 
	Note: Droplets are now being generated.</li>
	<li>Carefully and slowly transfer 40 &#956;L of the droplet-formed sample (top row) into separate wells of a 96-well plate by pipetting.</li>
	<li>After completing the droplet formation on the samples, seal the PCR plate with a pierceable dPCR foil at 180 &#176;C for 4 s.</li>
	<li>Place the foil-sealed PCR-plate in the cycler and cycle it as described in Table 4, at a ramp rate of 2.5 &#176;C/s.</li>
</ol>
4. Droplet Reading and Analyzing
<ol>
	<li>Transfer the plate from the thermal cycler to the base of the plate holder. Tighten the PCR plate by placing the top of the plate holder onto the PCR plate.</li>
	<li>Start the commercial dPCR software.</li>
	<li>Enter the sample name, the name of the experiment, and the target in the setup. Select the absolute quantification.</li>
	<li>Start the droplet reading by clicking RUN. Select FAM/HEX or FAM/VIC as detection chemistry when working with hydrolysis probes. 
	Note: The commercial dPCR software will then auto-analyze the data.</li>
	<li>Check the droplet numbers in the results table to ensure a correct droplet generation.</li>
	<li>Select all wells with the same quantified miRNA and click ANALYZE. Use the 2D blot to mark positive droplets and manually correct the auto-analyzed data (Figure 1).</li>
</ol>
5. Correct Calculations on the Sample
<ol>
	<li>Normalize the sample by multiplying the sample by the normalization factor (NF). 
	NF = Median [C. elegans miR-39 measurements]all samples/[C. elegans miR-39]given sample</li>
	<li>Calculate the final miRNA serum concentration, adjusting for dilution factors (DF). 
	[miRNA]final = [miRNA]raw value x DFRT x DFdPCR x DFEx 
	Where: 
	[miRNA]raw value = the miRNA quantified by commercial dPCR software; 
	DFRT = the dilution factor of the template in reverse transcription; 
	DFdPCR = the dilution factor of the template in dPCR; 
	DFEx = the dilution factor in the extraction.</li>
</ol>
6. Synthetic Oligonucleotide Dilution Series
<ol>
	<li>Thaw lyophilized synthetic oligonucleotide on ice. Briefly centrifuge it.</li>
	<li>Dilute the lyophilized synthetic oligonucleotide in nuclease-free water to a final concentration of 10 pmol/&#181;L. Briefly centrifuge it.</li>
	<li>Dilute it in nuclease-free water as described in Table 5. Centrifuge the dilution briefly between each dilution step at 8,000 x g for 10 s.</li>
	<li>Continue with the reverse transcription as described in step 2 and analyze the samples with digital PCR as described in step 3 and step 4.</li>
</ol>

<ol><li>Schulte, C., Zeller, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((microRNA-based+diagnostics+and+therapy+in+cardiovascular+disease+-+summing+up+the+facts%5BTitle%5D)+AND+%22Cardiovascular+Diagnosis+and+Therapy%22%5BJournal%5D)">microRNA-based diagnostics and therapy in cardiovascular disease - summing up the facts.</a> Cardiovascular Diagnosis and Therapy. 5, 17-36 (2015).</li>
<li>Sun, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+microRNAs+in+myocardial+infarction%3A+from+molecular+mechanism+to+clinical+application%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">The role of microRNAs in myocardial infarction: from molecular mechanism to clinical application.</a> International Journal of Molecular Sciences. 18, (2017).</li>
<li>Dimmeler, S., Zeiher, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+microRNAs%3A+novel+biomarkers+for+cardiovascular+diseases%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">Circulating microRNAs: novel biomarkers for cardiovascular diseases.</a> European Heart Journal. 31, 2705-2707 (2010).</li>
<li>Creemers, E. E., Tijsen, A. J., Pinto, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+microRNAs%3A+novel+biomarkers+and+extracellular+communicators+in+cardiovascular+disease%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease.</a> Circulation Research. 110, 483-495 (2012).</li>
<li>Oerlemans, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+assessment+of+acute+coronary+syndromes+in+the+emergency+department%3A+the+potential+diagnostic+value+of+circulating+microRNAs%5BTitle%5D)+AND+%22EMBO+Molecular+Medicine%22%5BJournal%5D)">Early assessment of acute coronary syndromes in the emergency department: the potential diagnostic value of circulating microRNAs.</a> EMBO Molecular Medicine. 4, 1176-1185 (2012).</li>
<li>Olivieri, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+potential+of+circulating+miR-499-5p+in+elderly+patients+with+acute+non+ST-elevation+myocardial+infarction%5BTitle%5D)+AND+%22Internation+Journal+of+Cardiology%22%5BJournal%5D)">Diagnostic potential of circulating miR-499-5p in elderly patients with acute non ST-elevation myocardial infarction.</a> Internation Journal of Cardiology. 167, 531-536 (2013).</li>
<li>Navickas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+circulating+microRNAs+as+biomarkers+of+cardiovascular+disease%3A+a+systematic+review%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Identifying circulating microRNAs as biomarkers of cardiovascular disease: a systematic review.</a> Cardiovascular Research. 111, 322-337 (2016).</li>
<li>Devaux, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+and+prognostic+value+of+circulating+microRNAs+in+patients+with+acute+chest+pain%5BTitle%5D)+AND+%22Journal+of+Internal+Medicine%22%5BJournal%5D)">Diagnostic and prognostic value of circulating microRNAs in patients with acute chest pain.</a> Journal of Internal Medicine. 277, 260-271 (2015).</li>
<li>Schwarzenbach, H., da Silva, A. M., Calin, G., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Data+normalization+strategies+for+microRNA+quantification%5BTitle%5D)+AND+%22Clinical+Chemistry%22%5BJournal%5D)">Data normalization strategies for microRNA quantification.</a> Clinical Chemistry. 61, 1333-1342 (2015).</li>
<li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Analytical Chemistry. 83, 8604-8610 (2011).</li>
<li>Hindson, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absolute+quantification+by+droplet+digital+PCR+versus+analog+real-time+PCR%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Absolute quantification by droplet digital PCR versus analog real-time PCR.</a> Nature Methods. 10, 1003-1005 (2013).</li>
<li>Robinson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Droplet+digital+PCR+as+a+novel+detection+method+for+quantifying+microRNAs+in+acute+myocardial+infarction%5BTitle%5D)+AND+%22Internation+Journal+of+Cardiology%22%5BJournal%5D)">Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction.</a> Internation Journal of Cardiology. 257, 247-254 (2018).</li>
<li>Mitchell, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+microRNAs+as+stable+blood-based+markers+for+cancer+detection%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Circulating microRNAs as stable blood-based markers for cancer detection.</a> Proceedings of the National Academy of Sciences of the United States of America. , 10513-10518 (2008).</li>
<li>D'Alessandra, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+microRNAs+are+new+and+sensitive+biomarkers+of+myocardial+infarction%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">Circulating microRNAs are new and sensitive biomarkers of myocardial infarction.</a> European Heart Journal. 31, 2765-2773 (2010).</li>
<li>Forootan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+to+determine+limit+of+detection+and+limit+of+quantification+in+quantitative+real-time+PCR+%28qPCR%29%5BTitle%5D)+AND+%22Biomolecular+Detection+and+Quantification%22%5BJournal%5D)">Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR).</a> Biomolecular Detection and Quantification. 12, 1-6 (2017).</li>
<li>Dingle, T. C., Sedlak, R. H., Cook, L., Jerome, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tolerance+of+droplet-digital+PCR+vs+real-time+quantitative+PCR+to+inhibitory+substances%5BTitle%5D)+AND+%22Clinical+Chemistry%22%5BJournal%5D)">Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances.</a> Clinical Chemistry. 59, 1670-1672 (2013).</li>
<li>Taylor, S. C., Laperriere, G., Germain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Droplet+digital+PCR+versus+qPCR+for+gene+expression+analysis+with+low+abundant+targets%3A+from+variable+nonsense+to+publication+quality+data%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data.</a> Scientific Reports. 7, 2409(2017).</li>
</ol>

The authors have nothing to disclose.

Digital PCR is a relatively novel end-point method of PCR that allows the direct absolute quantification of nucleic acids within a sample. The method possesses particular advantages, including a decreased variability, an increased day-to-day reproducibility, and a superior sensitivity11,12. Further, due to the partitioning of the sample into approximately 20,000 single reactions and endpoint analyses, dPCR is more robust to interfering substances in PCR compared ...

Circulating miRNAs have been identified as promising markers for a number of diseases, including cardiovascular disease1. The miRNAs are small, non-coding single-stranded RNA molecules (approximately 22 nucleotides long) are involved in the post-transcriptional regulation via the alteration of the messenger RNA translation and influencing gene expression2, and are released into the circulation in both physiological and pathological states. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity1. In cardiovascular diseases, miRNAs have become attractive candidates as novel biomarkers because they are remarkably stable in the serum and can easily be quantified with the help of PCR methodology3. The potential value of miRNAs as biomarkers for myocardial infarction has been evaluated in small studies, but a validation in large cohorts is lacking2. For example, miR-499 is found highly expressed in the myocardial muscle, and it has been shown to be significantly increased in an AMI4,5,6. Further, it regulates programmed cell death (apoptosis) and the differentiation of cardiomyocytes and is thus involved in several mechanisms following an AMI7. Apart from some small studies reporting a superiority and incremental value of miRNAs for the diagnosis of AMI, the superiority or equality to high-sensitivity cardiac troponins has not yet been proven in large-scale studies2,5,6,8. More prospective studies in large cohorts are, therefore, needed to assess the potential diagnostic value of miRNAs. Additionally, methods of miRNA quantification need to be optimized and standardized using comparable protocols9. Standardized assays may reduce inconsistent results and may help miRNAs to become potential biomarkers for the routine clinical application, as biomarkers need to be quantified in a reproducible way to ensure their clinical applicability.
Recently, dPCR has been introduced as an end-point analysis. It partitions the sample into approximately 20,000 individual reactions10. The dPCR system then utilizes a mathematical Poisson statistical analysis of fluorescent signals (positive and negative reactions), enabling an absolute quantification without a standard curve10. When combining dPCR with fluorescent hydrolysis probes, the highly specific direct absolute quantification of miRNAs is made possible. Digital polymerase chain reaction has shown to exhibit superior technical qualities (including a decreased variability, an increased day-to-day reproducibility, a high degree of linearity, and a high sensitivity) for quantifying miRNA levels in the circulation compared to quantitative real-time PCR10,11. These superior technical qualities might help to mitigate current limitations on using circulating miRNAs as biomarkers and may lead to the establishment of miRNAs as biomarkers in large multi-center cardiovascular clinical trials and as a diagnostic method in general. In a previous study, we recently applied dPCR for the absolute quantification of circulating miRNAs in patients with an AMI and were able to demonstrate superior diagnostic potential compared to the quantification of miRNAs by qPCR12.
In this publication, we want to demonstrate that using dPCR is an accurate and reproducible method for directly quantifying circulating cardiovascular miRNAs. The absolute quantification of miRNA levels in serum, using digital PCR, shows potential for the use in large multi-center cardiovascular clinical trials. In this publication, we describe in detail how to effectively perform digital PCR and how to detect the absolute miRNA copy number in serum.

<table><tbody><tr><td>&lt;strong&gt;RNA-Extraction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>miRNeasy Serum/Plasma Kit (50)</td><td>Qiagen-Sample &amp;amp; Assay Technologies, Hilden, Deutschland</td><td>217184</td><td>Kit for microRNA extraction. Kit contains commercial buffer RWT (called number one in the manuscript)&amp;nbsp; and RPE (called number two in manuscript).</td></tr><tr><td>miRNeasy Serum/Plasma Spike-in-Control; Syn-cel-miR-39 miRNA; 10pmol</td><td>Qiagen-Sample &amp;amp; Assay Technologies, Hilden, Deutschland</td><td>219610</td><td>Spike-in for normalisation , Sequence: 5&#039;-UCACCGGGUGUAAAUCAGCUUG-3&#039;</td></tr><tr><td>&lt;strong&gt;Reverse Transcription&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit (1000 Reactions)</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4366597</td><td>Kit for microRNA reverse transcription</td></tr><tr><td>TaqMan MicroRNA Assays M</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4440887</td><td>&lt;em&gt;Assays used in reverse transcription&lt;/em&gt;</td></tr><tr><td>hsa-miR-499 (750 RT/750 PCR rxns)</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4440887</td><td>Assay Number 001352</td></tr><tr><td>cel-miR-39 (750 RT/750 PCR rxns)</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4440887</td><td>Assay Number 000200</td></tr><tr><td>PCR Plate, 96-well, segmented, semi-skirted</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>AB0900</td><td>96 well plate for reverse transcription</td></tr><tr><td>Microseal &amp;lsquo;B&amp;rsquo; seal Seals</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>MSB1001</td><td>Foil to ensure proper storage</td></tr><tr><td>C1000 Touch Thermal Cycler</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1851196</td><td>Cycler used for reverse transcription</td></tr><tr><td>&lt;strong&gt;Droplet Digital PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 nmole RNA oligo hsa-miR-499-5p</td><td>Integrated DNA Technologies</td><td>Custom</td><td>Sequence: 5&#039;-phos-UUAAGACUUGCAGUGAUGUUU-3&#039;</td></tr><tr><td>ddPCR Supermix for Probes (No dUTP)</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863024</td><td>Supermix used in droplet generation</td></tr><tr><td>TaqMan MicroRNA Assays</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4440887</td><td>&lt;em&gt;Assays used in digital PCR (fluorescent hydrolysis probe)&lt;/em&gt;</td></tr><tr><td>hsa-miR-499 (750 RT/750 PCR rxns)</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4440887</td><td>Assay Number 001352, commercial primers</td></tr><tr><td>cel-miR-39 (750 RT/750 PCR rxns)</td><td>Applied Biosystems, Inc., Foster City, CA, USA</td><td>4440887</td><td>Assay Number 000200, commercial primers</td></tr><tr><td>DG8 Cartridges and Gaskets</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1864007</td><td>Cartridge takes up to 8 samples for droplet generation</td></tr><tr><td>DG8 Cartridge Holder</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863051</td><td>Holds cartridges in droplet generation</td></tr><tr><td>Droplet Generation Oil for Probes</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863005</td><td>Oil used in droplet generation</td></tr><tr><td>ddPCR 96-Well Plates</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>12001925</td><td>96 well plate for ddPCR</td></tr><tr><td>PCR Plate Heat Seal, foil, pierceable</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1814040</td><td>Pierceable foil, compatible with droplet reader</td></tr><tr><td>ddPCR Droplet Reader Oil</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863004</td><td>Oil used in droplet reading</td></tr><tr><td>QX100 or QX200 Droplet Generator</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863002</td><td>Droplet Generator, generates the droplets from sample/oil emulsion</td></tr><tr><td>PX1 PCR Plate Sealer</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1814000</td><td>Seals the plate before PCR</td></tr><tr><td>C1000 Touch Thermal Cycler</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1851196</td><td>Cycler used for ddPCR</td></tr><tr><td>QX100 or QX200 Droplet Reader</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863003</td><td>Reads PCR-positive and PCR-negative droplets with an optical detector</td></tr><tr><td>ddPCR Buffer Control for Probes</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1863052</td><td>Blank control&amp;nbsp;and to fill up the remaining wells of 8-well cassette</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>QuantaSof Software</td><td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td><td>1864011</td><td>Program for droplet reading</td></tr><tr><td>Prism Windows 5</td><td>GraphPad Software Inc., La Jolla, CA, USA</td><td></td><td>Program for statistical analysis</td></tr></tbody></table>

digital pcr for quantifying circulating micrornas in acute myocardial infarction and cardiovascular disease

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Digital PCR combined with fluorescent hydrolysis probes enables researchers to directly quantify the absolute amounts of specific miRNAs in copies/µL. As the sample in dPCR is partitioned in approximately 20,000 individual PCR reactions, dPCR does not require technical replicates10. The dPCR system utilizes a mathematical Poisson statistical analysis of fluorescent signals (differing between positive and negative reactions) enabling an absolute quantification witho...

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

digital-pcr-for-quantifying-circulating-micrornas-acute-myocardial

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MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Quantification of Circular RNAs Using Digital Droplet PCR

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Acute Myocardial Infarction in Rats

Learn digital PCR for quantifying circulating microRNAs as cardiovascular disease biomarkers using serum samples. This method enables precise miRNA measurement without standard curves for diagnostic research.

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

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