作者没有确认。

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。支持涡流的裂解。将管子与裂解液放在工作台的室温下5分钟。</li>
	<li>用3.5 µL 控制 (1.6 x 107拷贝/µL) 将裂解液钉在一起, 并用涡流混合。</li>
	<li>添加50µL 的氯仿 (即与起始血清样本相等的数量) 用于相分离。大力摇动管十五年代. 把管子放在工作台的室温下 2-3 分钟。</li>
	<li>为相分离, 离心机管在 1.2万 x g 在4°c 15 分钟。 注: 样品现在被分离成含 RNA 的上部水相, 白色相间, 和一个较低的, 粉红色的有机相。</li>
	<li>将含有 RNA 的上水相 (100 µL) 转移到新管中。不要转移任何白色相间的材料, 因为这伪造正确的 RNA 计数。</li>
	<li>添加150µL (即1.5x 的转移样品体积) 100% 乙醇。把这些材料吹打上下混合。不要将它们离心, 然后快速移动到下一步。</li>
	<li>将样品的250µL 在2毫升收集管的商用旋柱上。关上盖子。离心机柱在 > 8000 x g 在室温下十五年代. 丢弃流程。</li>
	<li>将700µL 的商用洗涤缓冲器编号1添加到自旋列。关闭盖子和离心机柱在 > 8000 x g 在室温下十五年代. 丢弃运行时的洗涤缓冲区。</li>
	<li>将500µL 的商用洗涤缓冲器编号2添加到自旋列。关闭盖子和离心机柱在 > 8, 000 x g 在室温下十五年代. 丢弃运行时的洗涤缓冲区。</li>
	<li>吸管500µL 80% 乙醇到自旋柱。关闭盖子, 离心机柱在 > 8, 000 x g 在室温下2分钟. 丢弃收集管与流经。</li>
	<li>将旋转柱转移到新的2毫升收集管。用打开的盖子将该柱离心在全速上晾干膜。将收集管丢弃, 并通过流经。</li>
	<li>将旋转柱转移到新的1.5 毫升收集管中。洗脱将30µL 的无 RNase 水直接应用于膜的中心。关上盖子。以全速离心柱1分钟。</li>
	<li>将 RNA 储存在-70 摄氏度。 注: 在-70 摄氏度至-20 摄氏度之间的 RNase 中纯化 rna 的储存确保 rna 的降解不超过1年。按照制造商的协议, 最低限度的 RNase 水稀释 RNA 是10µL. 使用少于10的µL 可能不足以使膜水化, 并降低总 RNA 的产量。</li>
</ol>2. 反向转录
注: 补充 DNA (cDNA) 是使用以下15µL 反向转录 (RT) 协议 (总反应量) 合成的。
<ol><li>在冰上解冻 RT 套件。尽可能长时间保持冷冻中的酶, 以防止它们在使用前降解和旋转。在冰上解冻 RT 底漆, 在使用前将其旋转。</li>
	<li>如表 1所述, 准备主组合。</li>
	<li>结合10µL 的主混合和5µL 提取 RNA 每井在96井板。</li>
	<li>离心机的井板在 2000 x g 在4°c 2 分钟。</li>
	<li>使用表 2中描述的热循环协议进行反向转录。 注: 如下所述, 可直接在数字 PCR 机中处理 cDNA (见材料表);或者, 它可以存储在摄氏-20 摄氏度。</li>
</ol>3. 液滴生成和数字 PCR
注: 使用以下40µL 协议进行了数字 PCR。
<ol><li>解冻的商业 dPCR 混合, 制备的 cDNA, 和 PCR 引物在冰上。在使用前不久将其离心。</li>
	<li>如表 3所述, 准备主组合。
</li>
	<li>添加 1.33 ul/井的 RT 产品和离心机简要。</li>
	<li>吸管 20 ul 样品入每井8井卡带 (中等列)。 注: 如果8井盒没有完全填充, 填补剩余的水井与非模板控制 (NTCs) 省略 cDNA (即RT 产品产生的水, 而不是提取 RNA) 或 dPCR 商业缓冲。NTC 作为污染控制。水不能在剩余的油井中使用, 因为这会损害液滴形成的质量。</li>
	<li>吸管 70 ul 的水滴产生油的探针进入油井 (底部的行) 的8井卡带。</li>
	<li>将垫片放在8井盒的顶部, 将卡带放在液滴发生器中。关闭液滴发生器。等待3指示灯呈稳定绿色。 注: 现在正在生成水滴。</li>
	<li>仔细和慢慢地转移 40 ul 的液滴形成的样品 (顶排) 成单独井的一个96井板由吹打。</li>
	<li>在样品上完成液滴形成后, 用 pierceable dPCR 箔在180摄氏度上密封 PCR 板4秒。</li>
	<li>将铝箔密封的 PCR 板放在循环仪中, 并按表 4所述循环, 以2.5 摄氏度/秒的坡道速率。</li>
</ol>4. 滴读和分析
<ol><li>将板从热循环仪转移到板架底座上。将板架顶部放在 pcr 盘上, 拧紧 pcr 盘。</li>
	<li>启动商业 dPCR 软件。</li>
	<li>在安装程序中输入示例名称、实验名称和目标。选择绝对量化。</li>
	<li>通过单击 "运行" 开始读取水滴。在使用水解探针时, 选择 "v" /"十六进制" 或 " VIC " 作为检测化学。 注意: 商业 dPCR 软件将自动分析数据。</li>
	<li>检查结果表中的水滴数以确保生成正确的雾滴。</li>
	<li>选择具有相同量化 miRNA 的所有井, 然后单击 "分析"。使用2D 印迹标记正滴, 并手动更正自动分析的数据 (图 1)。</li>
</ol>5. 对样品的正确计算
<ol><li>通过将样本乘以规范化因子 (NF) 来规范化样本。 NF = 中位 [线虫miR-39 测量]所有样品/[c. 线虫miR-39]给出样品
</li>
	<li>计算最终 miRNA 血清浓度, 调整稀释因子 (DF)。 miRNA最终= [miRNA]原始值x df x dfdPCR x dfEx 地方 miRNA原始价值= 商业 dPCR 软件量化的 miRNA; 在反向转录中模板的稀释因子; DFdPCR = 模板在 dPCR 中的稀释系数; DF = 稀释因子在萃取。</li>
</ol>6. 合成寡核苷酸稀释系列
<ol><li>解冻冻干合成寡核苷酸在冰上。简要地离心它。</li>
	<li>稀释核酸酶中的冻干合成寡核苷酸, 最终浓度为 10 pmol/µL. 简单地离心它。</li>
	<li>如表 5所述, 将其稀释在无核酸酶水中。离心稀释在每个稀释的步之间简要地在 8000 x g 十年代。</li>
	<li>按照步骤2中的说明继续进行反向转录, 并按照步骤3和步骤4中的说明对样本进行数字 PCR 分析。</li>
</ol>

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作者没有什么可透露的。

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>&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

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 ...

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

Research

JoVE Journal

Medicine

8.0K 次观看.  University of Freiburg. 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。

视频: 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.

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

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

科研

生物工程

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.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

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 ...

癌症研究

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.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

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

遗传学

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.

转移性结直肠癌患者循环微 rna 自定义面板的检测

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.

微RNA循环使用定量DNA结合染料化学和液滴的数字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 ...

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.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

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

生物学

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, ...

医学

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.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

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.

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

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.

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

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 ...

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

摘要

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高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

转移性结直肠癌患者循环微 rna 自定义面板的检测

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

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

基于探针实时荧光定量PCR途径的小分子RNA定量检测

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

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

摘要

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此视频中的章节

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

转移性结直肠癌患者循环微 rna 自定义面板的检测

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

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

基于探针实时荧光定量PCR途径的小分子RNA定量检测

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心