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

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1. 从血浆/血清中提取 miRNA
注意: 为了适当量化 miRNAs, 正确的 microRNA 隔离从血浆/血清是一个关键的步骤。要记住的关键一点是, 特别是由于存在不同的协议, 在处理示例时要遵循相同的工作流。在本议定书中, miRNA 从50µL 的血清中提取。不要使用超过200µL, 因为这限制了正确的提取过程。
<ol><li>准备血清或解冻冷冻样品。</li>
	<li>添加250µL (5 卷) 商业裂解试剂到50µL 血清。支持...

<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>

数字 pcr 是一种相对新颖的 pcr 终点法, 它允许在样品中直接对核酸进行绝对量化。该方法具有特殊的优点, 包括可变性降低, 每日重现性增加, 灵敏度11,12。进一步, 由于样品分成大约2万个单一反应和端点分析, dPCR 比定量 rt-pcr16更健壮对干扰物质在 pcr。这些品质在 dPCR 使它成为一个有吸引力的替代定量 rt-pcr 作为诊断工具的量化。由于?...

循环 miRNAs 已被确定为许多疾病, 包括心血管疾病1的有前景的标志。miRNAs 是小的, 非编码单链 RNA 分子 (大约22核苷酸长)通过信使 RNA 翻译的改变和影响基因表达2参与后转录调控,并被释放到循环中的生理和病理状态。具体 miRNAs 的定量表达可以与病理学相联系, 一些 miRNAs 表现出较高的组织和疾病特异性1。在心血管疾病中, miRNAs 已成为具有吸引力的候选生物标志物, 因为它们在血清中明显稳定, 可以在 PCR 方法3的帮助下很容易量化。miRNAs 作为心肌梗死的生物标志物的潜在价值在小的研究中得到了评价, 但在大群群中的验证却缺乏2。例如, miR-499 被发现在心肌肌肉中表现得很高, 在 AMI4、5、6中表现出明显的增加。此外, 它调节程序性细胞死亡 (凋亡) 和心肌细胞的分化, 因而参与了 AMI7后的几个机制。除了一些小的研究报告 miRNAs 的优势和增量价值的诊断 AMI, 高灵敏度的心脏肌钙蛋白的优越性或平等尚未证明在大规模研究2,5 ,6,8。因此, 需要对大型群体进行更多前瞻性研究, 以评估 miRNAs 的潜在诊断价值。此外, miRNA 量化的方法需要优化和标准化使用可比较的协议9。标准化化验可以减少不一致的结果, 并可能有助于 miRNAs 成为潜在的生物标志物的常规临床应用, 因为生物标志物需要量化的可重复的方式, 以确保其临床适用性。
最近, dPCR 作为一个终点分析被引入。它划分样品入大约2万个各自的反应10。dPCR 系统然后利用数学泊松统计分析的荧光信号 (正负反应), 使绝对量化没有标准曲线10。将 dPCR 与荧光水解探针相结合, 使 miRNAs 的高度特异直接绝对量化成为可能。数字聚合酶链反应显示出优越的技术品质 (包括可变性下降, 日到日重现, 高度线性度和高灵敏度), 以量化 miRNA 水平在循环比定量实时 PCR10,11。这些优越的技术品质可能有助于减轻目前使用循环 miRNAs 作为生物标志物的限制, 并可能导致在大型多中心心血管临床试验中建立 miRNAs 作为生物标记, 并作为一种诊断方法在一般。在先前的一项研究中, 我们最近应用 dPCR 对 AMI 患者循环 miRNAs 的绝对量化, 并能显示出优于 qPCR12定量 miRNAs 的诊断潜力。
在本刊物中, 我们想证明使用 dPCR 是一种准确和可重复的方法直接量化循环心血管 miRNAs。用数字 PCR 技术对血清中 miRNA 水平的绝对量化, 显示了在大型多中心心血管临床试验中的应用潜力。在本论文中, 我们详细描述了如何有效地执行数字 PCR 以及如何检测血清中的绝对 miRNA 拷贝数。

<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

循环血清 microRNAs (miRNAs) 表明, 作为心血管疾病和急性心肌梗死 (AMI) 的标志物, 从心血管细胞释放到循环。循环 miRNAs 高度稳定, 可量化。具体 miRNAs 的定量表达可以与病理学相联系, 一些 miRNAs 表现出较高的组织和疾病特异性。为心血管疾病寻找新的生物标志物对医学研究具有重要意义。最近, 数字聚合酶链反应 (dPCR) 已经发明。dPCR, 结合荧光水解探针, 使特定的直接绝对量化。与定量聚合酶链反应 (qPCR) 相比, dPCR 具有优异的技术质量, 包括低变异性、高线性度和高灵敏度。因此, dPCR 是一种更准确和可重复的方法直接量化 miRNAs, 特别是用于大型多中心心血管临床试验。在本论文中, 我们描述了如何有效地执行数字 PCR, 以评估血清样本中的绝对拷贝数。

数字 PCR 结合荧光水解探针使研究人员可以直接量化的绝对数量的特定 miRNAs 在拷贝/µL。当样品在 dPCR 被划分在大约2万个单独 PCR 反应, dPCR 不需要技术复制10。dPCR 系统利用数学泊松统计分析的荧光信号 (不同的正面和负面的反应), 使绝对量化, 而无需标准曲线10。为了正确地计算浓度, 需要一个可接受的水滴计数 (> 1.5万)。没有模板控件?...

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

循环 microRNAs 表明, 作为心血管疾病和急性心肌梗死的标志物的承诺。在本研究中, 我们描述了一个 miRNA 提取, 反向转录和数字 PCR 的协议, 以绝对量化的 miRNAs 在心血管疾病患者的血清。

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

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

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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

研究小鼠心肌梗死的低温损伤模型

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

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

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

Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植

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