Name: digital pcr for quantifying circulating micrornas in acute myocardial infarction and cardiovascular disease
Uploaded: 2018-07-03T14:45:00.000+00:00
Duration: 4 min 41 s
Description: Watch this Scientific Journal Video about Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease at JoVE.com

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. 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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

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 ...

Watch this Scientific Journal Video about Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease at JoVE.com

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 ...

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

Research

JoVE Journal

Medicine

8.1K Views.  University of Freiburg. Circulerende microRNAs is gebleken belofte als biomarkers voor cardiovasculaire aandoeningen en acuut myocardiaal infarcten. In deze studie beschrijven we een protocol voor extractie van miRNA, omgekeerde transcriptie en digitale PCR voor de absolute kwantificering van miRNAs in het serum van patiënten met hart-en vaatziekten.

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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Cancer Research

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

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

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

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.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Genetics

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

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

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.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Biology

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

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

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

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.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

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

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

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.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

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

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