Name: circulating microrna quantification using dna-binding dye chemistry and droplet digital pcr
Uploaded: 2016-06-26T14:00:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR at JoVE.com

从意大利癌症研究协会（AIRC）为MF（MFAG 11676）和MN资金支持（特别节目分子临床肿瘤学 - 5千分数ñ9980，2010/15）和指令意大利教育部，大学和研究FIRB 2011年明尼苏达州（项目RBAPIIBYNP）。

从血浆或血清中分离的microRNA 注意:血浆和血清制剂是循环的miRNA定量的相关步骤。存在用于血浆和血清制备没有优选的程序。唯一要考虑的重要的事情是，所有来自同一实验的样品必须使用完全相同的工作流程进行处理。从200μl的血清或血浆开始。总RNA可以从血清或血浆用市售试剂盒进行分离。 1，协议总RNA（miRNA的包括）隔离<ol><li>在冰上解冻血清/血浆样本。 </li><li> 1毫升裂解试剂（ 如 ，Qiazol加）添加到200微升血清/血浆。 </li><li>涡旋混匀，含在板凳上的匀浆在室温5分钟管。 </li><li>从添加C的合成的miRNA CEL-MIR-39-3p的4.16纳米的解决方案的3微升线虫 。 </li><li>加入200μl氯仿。摇动该管剧烈持续15秒。第广场在室温替补2分钟就电子管。 </li><li>离心机以12,000 xg离心15分钟，在4℃，以获得相分离:上层水相含有的RNA。 </li><li>水相转移到一个新的管，约700微升，避免任何白色相间的材料转移。 </li><li>加入1ml的100％乙醇并通过倒置混合。 </li><li>吸管向上700微升样品放入一个微型旋转柱置于2ml的收集管。盖上盖子，离心机在12000 XG为15秒。丢弃的流量通过。 </li><li>重复使用剩余的样本这一步骤。 </li><li>加入700μlBuffer RWT到微型离心柱，盖上盖子，离心15秒12,000 XG洗列。丢弃的流量通过。 </li><li>吸取500微升缓冲液RPE到微型离心柱。盖上盖子，离心15秒12,000 RPM。丢弃的流量通过。再次重复此步骤。 </li><li>离心全速微型旋转柱2分钟以干燥离心柱从膜残留的乙醇。 </li><li>微型旋转柱转移至新的1.5 ml收集管和移液管35微升不含RNA酶的水在列的膜。 </li><li>离心1分钟以12,000 xg离心以洗脱RNA。 </li><li>储存在-80℃的RNA。 注意:由于RNA浓度不能精确确定的，使用固定的卷作为输入量的量度。 </li></ol> 2.微小RNA反转录<ol><li>解冻的逆转录（RT）试剂混合物成分:5×反应缓冲液，不含核酸酶的水。混合轻轻翻转冰上管和地点。在使用前，立即删除从冰箱中的酶混合物置于冰上。降速所有试剂。 </li><li>如表1中所述制备在冰上对RT试剂混合物。制备至少10％的超过组合。 </li><li>混合吹打反应，然后降速。 </li><li>在42℃孵育60分钟。 </li><li>热灭活的逆向工程＆本身酶为在95℃5分钟。 </li><li>立即冷却至4℃。 </li><li>存放在-20°C的基因。 </li></ol> 3.稀释的cDNA <ol><li>稀需要在无核酸酶的水计划ddPCR反应cDNA模板的量。稀释1:50和1之间的基因反应:500水，取决于目标miRNA的丰度。储存在-20℃下的稀释的cDNA。所述稀释的cDNA被证明为在-20℃至少3个月是稳定的。 </li></ol> 4.产生液滴和PCR 注意:液滴生成应该在8个样品在同一时间内进行。技术重复不是必需的，因为这种技术12,15 .A无模板对照（NTC）的样品应当在每一个板中运行的高再现性，并为每个不同ddPCR条件。 <ol><li>解冻并平衡了主结构（ 例如 ，EvaGreen预混），微小RNA引物和cDNA在室温。 </li><li>混合母液日oroughly通过倒转试管几次。降速所有试剂。 </li><li>如表2中所述制备ddPCR组合。当执行多个ddPCR反应，制备ddPCR混合工作溶液。至少准备10％的超额组合。 </li><li>一旦组装，调匀和降速ddPCR组合。 </li><li>分配12微升ddPCR的混合到96孔PCR板或PCR管。 </li><li>添加8微升稀释的cDNA模板的每个管/孔。 </li><li>将液滴生成墨盒插入支架。 </li><li>转移20微升每种制备的样品，以微滴发生器盒小心，同时避免空气在井的底部的气泡的样品孔（中间行）。 </li><li>用70微升液滴生成油填充每个油井（下排）。 </li><li>钩在盒保持器的垫片和插入液滴发生器。 </li><li>盖上盖子，开始根据液滴代制造商的公关otocol。当液滴的一代已经完成，打开盖子，取出一次性垫圈。盒的顶部孔包含液滴。 </li><li>吸管慢慢，平稳40微升的八个顶部孔（液滴）的内容到一个96孔PCR板中的一列。 </li><li>转移液滴蒸发避免后立即密封带箔的PCR板。使用可刺穿箔片密封件是与液滴读者针相容。 </li><li>密封板30分钟内开始热循环（PCR）。 </li><li>根据表3进行循环协议。 </li></ol> 5.阅读液滴<ol><li>上电液滴读者。 </li><li>从热循环仪，以液滴读者移动板。 </li><li>放置含有PCR后液滴入盘保持器的底部的96孔PCR板。 </li><li>放置在PCR板的板保持器的顶部。用力按下两个释放卡舌到固定支架中的PCR板。 </li><li>开始从系统计算机软件。 <ol><li>单击设置来定义试​​验（绝对定量），然后单击Run启动液滴读数。 </li><li>当液滴阅读完毕后，单击"分析"按钮，打开和分析数据。 </li><li>使用2D振幅积在分析软件来选择阳性微滴（套索工具）（ 图1B）。 </li><li>使用事件选项卡，检查阳性，总滴数。为18,000的总人数 - 21000飞沫通常与probeless ddPCR实现。 </li><li>一旦正液滴被选择，导出使用来自浓度标签导出的.csv选项所述miRNA浓度值。 </li></ol></li></ol>

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The authors have nothing to disclose.

循环miRNA是存在于血液中的浓度极低，而且可以从血浆和血清样品中提取的RNA的量是低的。因为这个原因，它们难以与其它技术，如微阵列和RNA测序量化。此外，还有在数据归一化的一般化缺乏协议和内源性"参考"微RNA在血液中的存在。在这种情况下，一个灵敏的技术，如微滴数字PCR，能够每毫升血清或血浆，即使非常低的计数的miRNA拷贝数的，是非常有吸引力的。我们配对该技术具有普遍性的cDNA系统，反向转录在一个反应​​全部循环的miRNA从而节省时间，最重要的是，RNA的材料。在我们的方法，特异度为固有的使用LNA的引物，相对于其他的解决方案时，16 miRNA的定量其中表现非常出色。 液滴数字PCR是终点测定法，允许靶基因的计算绝对浓度。不像定量PCR，扩增效率不足不影响最终ddPCR定量。因此，考虑到本比如血液样本中的PCR抑制剂的量，ddPCR提供用于循环核酸评估一个强大的工具。 由于其高灵敏度，该技术提供了一个足够的量化也不太丰富的miRNA，即使起始原料的量有限。另外，假设每个miRNA分子在cDNA分子反转录，我们可以计算出在1微升血浆/血清的绝对拷贝ddPCR反应值所得到的浓度乘以要稀释因子（145.83）。 所提出的工作流程可以在8-96的样品在一个时间容易地进行，从而提供了在CLINICA微RNA定量的可靠和时间有效的工具L个样本。滴数字PCR有可能成为核酸的一个必不可少的工具生物标志物评估，诊断设置，从而使液体活检从替补到床边的潜力。

MicroRNAs (miRNAs) are released into blood circulation by potentially all the cells of the organism, as a consequence of active release or necrotic and apoptotic processes. Cell-free miRNAs have been detected in the bloodstream either as free stable molecules or linked to lipoproteins or enveloped inside exosomes and microvesicles 1-3. They are believed to function as cell-to-cell communicators 4, and their amount changes in the presence of cancer, cardiac disorders or autoimmune diseases 5-7. Their accurate and reproducible quantification is the basis for their evaluation as disease biomarkers. However, for several reasons already described elsewhere 8,9, miRNA quantification in serum or plasma, as well as other body fluids, could be very challenging 10,11. We recently developed a method for the absolute quantification of circulating miRNAs, based on miRNA-specific LNA primers and DNA-binding dye droplet digital PCR (ddPCR) technology 12. This methodology has been applied to the validation of miRNA breast cancer biomarkers 13,14. 
After the partitioning of each reverse-transcribed miRNA molecule inside a nanoliter-sized droplet, it is possible to count the copy number of each miRNA in each sample, basically counting the number of green, and therefore positive, fluorescent droplets. As soon as a PCR reaction occurs, a positive count is achieved, without the need to establish a standard curve or taking PCR efficiency into account in target amount calculation, as it happens with quantitative RT-PCR (RT-qPCR). In addition, ddPCR proved to be more sensitive and accurate than RT-qPCR in circulating miRNA quantification 15. In this article we present the detailed protocol of this methodology, discussing the most relevant steps andspecifically considering serum and plasma clinical samples.

<table><tbody><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td>Columns for total RNA, including miRNA, extraction from serum/plasma</td></tr><tr><td>100 nmole RNA oligo Cel-miR-39-3p</td><td>Integrated DNA Technologies</td><td>Custom</td><td>Sequence: UCACCGGGUGUAAAUCAGCUUG</td></tr><tr><td>Universal cDNA synthesis kit II, 8-64 rxns</td><td>Exiqon</td><td>203301</td><td>Kit for microRNA reverse transcription</td></tr><tr><td>MicroRNA LNA PCR primer set&amp;nbsp;</td><td>Exiqon</td><td>204000-206xxx and 2100000-21xxxxx</td><td>Primers for miRNA amplification inside droplets</td></tr><tr><td>QX200 droplet generator</td><td>BioRad</td><td>186-4002</td><td>Instrument used for droplet reading</td></tr><tr><td>QX200 droplet reader</td><td>BioRad</td><td>186-4003</td><td>Instrument used for droplet generation</td></tr><tr><td>QuantaSoft software</td><td>BioRad</td><td>186-3007</td><td>Software for data collection and analysis</td></tr><tr><td>PX1 PCR plate sealer</td><td>BioRad</td><td>181-4000</td><td>Plate sealer</td></tr><tr><td>DG8 droplet generator cartridges and gaskets</td><td>BioRad</td><td>186-4008</td><td>Cartridges used to mix sample and oil to generate droplets</td></tr><tr><td>QX200 ddPCR EvaGreen supermix</td><td>BioRad</td><td>186-4033/36</td><td>PCR supermix</td></tr><tr><td>QX200 droplet generator oil for EvaGreen dye</td><td>BioRad</td><td>186-4005</td><td>Oil for droplet generation</td></tr></tbody></table>

circulating microrna quantification using dna-binding dye chemistry and droplet digital pcr

循环微RNA（miRNA）（无细胞的），从细胞释放到血流中。在循环的特定微小RNA的量已与一种疾病状态，并具有被用作疾病的生物标志物的潜力。用于使用基于染料的化学和液滴数字PCR技术循环微RNA定量的灵敏和准确的方法最近已开发。具体而言，使用锁核酸（LNA）为基础的miRNA特异性引物用绿色荧光DNA结合染料在相容的液滴数字PCR系统能够获得特定miRNA的绝对定量。在这里，我们描述了如何执行此技术，以评估在生物流体，例如血浆和血清的miRNA量，是可行和有效的。

可使用绿色荧光DNA结合染料和液滴数字PCR技术来确定每毫升的血浆或血清的特定miRNA的绝对量。 图1表示正极的液滴选择的过程中，它决定了最终的miRNA浓度（拷贝/微升）在由分析软件计算的扩增反应。血液中的每个miRNA的量是非常不同的，是一些miRNA族比其他人更丰富。使用在ddPCR反应1:50稀释的cDNA，可以得到正和负的液滴的足够数量。在正液滴饱和（ 例如 ，没有剩余的负液滴）的情况下，cDNA样本的进一步的稀释可能是必要的。 每一LNA的引物组应该被优化以液滴数字PCR系统上正常工作。对的miR-181A-5P优化过程是在图2中表示的具体而言，改变引物的量（通常在0.25的范围内-每ddPCR反应1微升）。和退火温度（通常在56的范围内- 60℃），它是可以选择的组合确定正和负的液滴之间的更好的分离。在的miR-181A-5P，60℃的退火温度和两个0.5和1μl的引物的量的情况下提供以液滴振幅而言更好的结果。 是可能发生的一组引物给出了一些非特异扩增，可能是由于引物二聚体的形成。当与循环的miRNA工作，从样品的正信号可能是非常低的，因此类似的非特异性信号。如果非特 ​​异性靶扩增的"云"不与真实的信号（ 图1C）重叠，所述miRNA仍可定量仅选择所述真实<html>积极的液滴。在这种情况下，NTC样品为液滴选择是必不可少的。所有被包含在相同的分析样品需要使用同样的方法和相同的ddPCR条件进行处理。 <img alt="图1" src="/files/ftp_upload/54102/54102fig1.jpg" /> 图1.积极液滴选择 。阳性液滴使用1D曲线（A）或执行围绕在二维图（B）中的正液滴云圆负液滴高于阈值的分析过程中手动选择。如果一些引物组给出了非特异性扩增，这是通过正液滴的第二云出现在阴性对照（无模板对照，NTC）样品（C）的证明。 "真正的"阳性仍然可以选择不包括在二维图中的非特异性信号。e.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54102/54102fig2.jpg" /> 图2.引物优化无细胞的miRNA定量与Probeless ddPCR上部面板:使用4种不同的ddPCR条件的两个血浆样品（S1和S2）中的miR-181A-5P的定量。 MIR-181A-5P LNA的引物浓度（0.5和1微升）和退火温度（58和60℃）影响的正（蓝色）中的DNA-嵌入染料ddPCR的振幅和负（黑色）的液滴。可以得到在60℃下进行PCR，并使用0.5或1μl的引物对本miRNA的更好的分离。对照样品（NTC，无模板对照）没有正面滴，符合市场预期。中央板:在上述条件下的miR-181-5p的最终浓度。结果表示为在放大器每微升份fication反应。误差棒代表泊松95％置信区间。下图:上液滴（绿色）的总人数正滴（蓝色）的数量<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54102/54102fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 体积（微升） </td></tr><tr><td> 5X反应缓冲液</td><td> 4 </td></tr><tr><td>不含核酸酶的水</td><td> 11 </td></tr><tr><td>酶混合物</td><td> 2 </td></tr><tr><td>模板总RNA </td><td> 3 </td></tr><tr><td> 总成交量 </td><td> 20 </td></tr></tbody></table> 表1 - 逆转录试剂混合组件 <table fo:keep-together.within-p年龄="1"&gt; <tbody><tr><td> 试剂 </td><td> 体积（微升） </td></tr><tr><td> 2X DNA结合染料超混合液</td><td> 10 </td></tr><tr><td> LNA引物</td><td>可变（0.5 - 1）* </td></tr><tr><td>不含核酸酶的水</td><td>变量</td></tr><tr><td>稀释cDNA模板</td><td> 8 </td></tr><tr><td> 总成交量 </td><td> 20 </td></tr><tr><td></td><td></td></tr><tr><td colspan="2"> *第一ddPCR反应应该是几样执行，建立底漆的最佳工作数量</td></tr></tbody></table> 表2 - ddPCR试 ​​剂混合组件 <table fo:keep-together.within-page="1"><tbody><tr><td> 循环步骤 </td><td> 温度 </td><td> 时间</td><td> 下降率 </td><td> 周期 </td></tr><tr><td>激活酶</td><td> 95℃ </td><td> 5分钟</td><td rowspan="6"> 〜2℃/秒</td><td> 1 </td></tr><tr><td>变性</td><td> 95℃ </td><td> 30秒</td><td rowspan="2"> 40 </td></tr><tr><td>退火/延伸</td><td> 60℃ </td><td> 1分钟</td></tr><tr><td rowspan="2">信号稳定</td><td> 4℃ </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 90℃ </td><td> 5分钟</td><td> 1 </td></tr><tr><td>保持（可选） </td><td> 4℃ </td><td>无穷</td><td> 1 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">使用加热的盖设置为105℃，样品体积为40微升。 </td><td></td></tr></tbody></table>表3 - 热循环条件液滴的数字PCR

Watch this Scientific Journal Video about Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR at JoVE.com

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

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 ...

circulating-microrna-quantification-using-dna-binding-dye-chemistry

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.6K 次观看.  University of Bologna. A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

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

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 and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

癌症研究

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

生物化学

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.

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.

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 ...

医学

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

生物学

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 ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

使用数字液滴聚合酶链反应测量单细胞线粒体DNA拷贝数和异质性

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

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 ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

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

使用数字微滴PCR定量环状RNA

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

摘要

探索更多视频

此视频中的章节

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

患者生物流体中肿瘤相关循环DNA的检测与监测

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

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

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

数字核酸定量实验分析的简单堆积读数

使用数字液滴聚合酶链反应测量单细胞线粒体DNA拷贝数和异质性

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

使用数字微滴PCR定量环状RNA

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

摘要

探索更多视频

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

患者生物流体中肿瘤相关循环DNA的检测与监测

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

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

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

数字核酸定量实验分析的简单堆积读数

使用数字液滴聚合酶链反应测量单细胞线粒体DNA拷贝数和异质性

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

使用数字微滴PCR定量环状RNA

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