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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-based+diagnostics+and+therapy+in+cardiovascular+disease+-+summing+up+the+facts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microRNAs+in+myocardial+infarction:+from+molecular+mechanism+to+clinical+application.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+novel+biomarkers+for+cardiovascular+diseases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+novel+biomarkers+and+extracellular+communicators+in+cardiovascular+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+assessment+of+acute+coronary+syndromes+in+the+emergency+department:+the+potential+diagnostic+value+of+circulating+microRNAs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+potential+of+circulating+miR-499-5p+in+elderly+patients+with+acute+non+ST-elevation+myocardial+infarction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+circulating+microRNAs+as+biomarkers+of+cardiovascular+disease:+a+systematic+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+prognostic+value+of+circulating+microRNAs+in+patients+with+acute+chest+pain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+normalization+strategies+for+microRNA+quantification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantification+by+droplet+digital+PCR+versus+analog+real-time+PCR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+as+a+novel+detection+method+for+quantifying+microRNAs+in+acute+myocardial+infarction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+stable+blood-based+markers+for+cancer+detection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+are+new+and+sensitive+biomarkers+of+myocardial+infarction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+determine+limit+of+detection+and+limit+of+quantification+in+quantitative+real-time+PCR+(qPCR).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+of+droplet-digital+PCR+vs+real-time+quantitative+PCR+to+inhibitory+substances.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+versus+qPCR+for+gene+expression+analysis+with+low+abundant+targets:+from+variable+nonsense+to+publication+quality+data.">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>RNA-Extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Kit (50)</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>217184</td>
			<td>Kit for microRNA extraction. Kit contains commercial buffer RWT (called number one in the manuscript)&#160; 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 &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>219610</td>
			<td>Spike-in for normalisation , Sequence: 5&#39;-UCACCGGGUGUAAAUCAGCUUG-3&#39;</td>
		</tr>
		<tr>
			<td>Reverse Transcription</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>Assays used in reverse transcription</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 &#8216;B&#8217; 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>Droplet Digital PCR</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&#39;-phos-UUAAGACUUGCAGUGAUGUUU-3&#39;</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>Assays used in digital PCR (fluorescent hydrolysis probe)</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&#160;and to fill up the remaining wells of 8-well cassette</td>
		</tr>
		<tr>
			<td>Software</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

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/&#181;L. 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 ...

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

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 ...

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8.0K Views.  University of Freiburg. 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.

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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

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

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...