这项工作是由美国国立卫生研究院拨款DE018304，R01DE018281支持。 KT公司是由T32 - 34 GM07092授予教堂山北卡罗来纳大学霍华德休斯医学研究所（HHMI）的通过MED到毕业倡议的支持。 PC支持的T32 CA009156。

qPCR的预miRNA表达谱阵列，可以成立一个特餐自由埃沃机器人（A）或手与基体的电子多道移液器（二）充分自动化。 1）准备主结构，引板和样品。 <ol><li>底漆板含有186个引物对96孔板格式在下午12时05分（共2板），应储存在-80 ° C解冻板在室温下，旋涡和离心机使用前简要。 </li><li>要准备的主结构，SYBR绿色2X PCR混合物在室温下解冻。每个反应使用2ul底漆8ul预混。每个反应的主结构的组成是4UL SYBR组合，3ul PCR级水和10-20吴样本DNA或cDNA。 </li><li>每96底漆板的设置，四大高手混合管是必要的。样品可以运行4个样本（singlicate）每盘，每盘2个样本（复印件）或单个样品一式四份。 </li><li>准备42毫升Eppendorf管相结合的SYBR，水和样品的主结构。每个管应包含足够的预混约100反应，让多余的废物吹打。涡旋混合。 </li></ol> A.安装前的miRNA使用自由特餐埃沃机器人的实验。 <ol><li>机器人和加载的Evoware程序初始化后，30mls每个冲洗三次，以清除气泡会干扰移液精度系统。 </li><li>安装的机器人平台，包括以下内容: <ul><li>标记384孔板</li><li> 96孔引物板（1） </li><li>主混音</li><li>含有2％的漂白粉低谷</li><li>一个充满制度液容器</li><li>空废物容器</li></ul></li><li>首先，选择"运行"两次自动机器人运行。 </li><li>在节目结束时，删除的LightCycler 480密封箔384孔板和密封，并短暂离心​​。酒店的位置1处密封384孔板。 </li><li>转到步骤2.2和底漆板2，与新的主混合和一个新的384孔板的重复。 <ol><li>密封板放置在酒店的位置2。 </li></ol></li><li>打开新Evoware程序加载从酒店在LightCycler，并选择"运行"两次。 <ol><li>自由埃沃将自动加载到每个板块的LightCycler，并运行以下的SYBR绿色的I /人力资源管理循环程序: 前（1个周期）: 50 ° 5分钟斜率4.8 ° /秒 95 ° 5分钟斜率4.8 ° /秒 放大器（45个循环）: 15秒95 °斜率4.8 ° /秒 30秒62 °斜率2.5 ° /秒 （在此步骤中的单个数据采集） 熔点曲线: 5秒，95 °斜率4.8 ° /秒 60℃1分钟斜率2.5 ° /秒 95 °持续升温速率为0.11 ° /秒 5收购每° C 酷: 30秒50 °斜率25 ° /秒</li></ol></li></ol> B.安装前miRNA的检测使用矩阵电子多道移液器: <ol><li>预混1到水库管放置的内容。 </li><li>设置电子移液器吸16ul 2 - 8ul预混免除吹扫步骤遵循的步骤。 </li><li>对于预混1，分配到每一个其他的384孔板（384孔尖间距设置）以及8ul，孔A1开始。 （即孔A1，C1，E1等），对于第二8ul免除，移动到第3列（即井A3，C3，E3，等），然后通过一个废物容器的清除和处理技巧。 </li><li>其余5个周期，按照这种模式，直到您到达以及A23。 </li><li>重复的master mix 2，（开头以及A2，C2，E2，等等）;预混3（孔B1，D1，F1等开始）;预混4（开始在B2，D2，F2键等。） </li><li>对于引物板aliquotting，设置电子移液器吸2ul并免除2ul与清除步骤如下。到384孔板，孔A1，C1，E1等在一个废物容器的清除和处理技巧，免除从96孔底漆板（AH）的第1列的引物2ul。重复井A2，C2，E2，B1，D1，F1;和B2，D2，F2键等。 </li><li>重复2-12行96引物，引物各列，每2列移动，直到全部384孔板已aliquotted。 </li><li>与LightCycler配套的密封铝箔和离心密封板。 </li><li>进入一个的LightCycler 480和周期的板块，按照以下设置使用SYBR GREEN I /人力资源管理检测格式板: 前（1个周期）: 50 ° 5分钟斜率4.8 ° /秒 95 ° 5分钟斜率4.8 ° /秒 安培（45个循环）: 在升温速率为15秒95 °4.8 ° /秒 30秒62 °斜率2.5 ° /秒 （在此步骤中的单个数据采集） 熔点曲线: 5秒95 °斜率4.8 ° /秒 60℃1分钟斜率2.5 ° /秒 95 °持续升温速率为0.11 ° /秒 5收购每° C 酷: 30秒50 °斜率25 ° /秒</li><li>重复使用底漆板2，使用新鲜配制的主混合到一个新的384盘aliquotting。 </li></ol>成功秘诀: 设计PCR扩增阵列时的一个重要特点是，所有186个引物对具有相同的退火温度，使板可运行在同样的最佳PCR运行的程序设置。 使用三个控件在运行样品前，应检查每一个新的数组。这些控制包括: 1运行与运行，以排除引物污染的水或0.1 x TE所有引物。 运行交替水/ TE和阳性对照，以确保无交叉污染。 3运行使用相同的阳性对照，以确保运行之间的可重复性。 主混音应新鲜配制，板应在24小时内运行，以获得最佳效果 应去除引物板箔，慢慢井间的污染，以减少风险。 在使用机器人与固定提示时，2％的漂白粉洗，洗彼此之间移液步骤的提示，通过用水冲洗是必要的。这样可以防止和消除结转，否则可能污染数据，导致不确定的结果。 后板是通过在LightCycler上运行，它不应该再次被打开的PCR设置房间。这有助于防止污染的PCR。 代表性的成果: qPCR的结果通常是代表在LightCycler分析软件确定CT值。通常包括积极为样本，样品，通常介于20-35的CT范围的成功运行。水样在一个良好的运行总是在&gt; 40 CT和任何样品或与CT的具体井超过37被认为是负面的，或者未检出。作为一般规则，样品产生&lt;10 CT也是不可靠的，从进一步的分析排除。有几种方法，来分析我们的检测的qPCR数据和内部控制纳入有助于控制样本输入的变化。预MIR阵列包括一个U6对照引物，这往往是用来作为参考基因正常化从不同样品的CT值。这被称为三角洲CT值（DCT）的标准化值。结果往往是绘制为原料的CT，DCT值或标准值可进一步分析。 在图1中的全部四个不同的样品从timecourse实验前的microRNA阵列分析热图格式。每个前的小分子RNA的相对表达显示，并出现了三大鲜明的集群。大多数的分析预大鹏进行了小的，微不足道的变化，如预期（图1A）。然而，前的小分子RNA的一小部分有显着下降（图1B）或整个timecourse实验的急剧增加（图1C）。 0H时间点（样品1）作为基线水平的每一个预选的microRNA的表达水平正常化。在某些情况下，您可能会注意到，一些聚集前的microRNA也集中在热图分析（例如:让7A，图1B）。根据实验，集群可能出现依赖于病毒感染的细胞类型前的小分子RNA或预大鹏的具体体现，是一个共同的分子信号转导通路调节。 我们也已经开发出包括前的小分子RNA检测已知病毒前- microRNA和卡波济氏肉瘤相关疱疹病毒（KSHV）和EB病毒（EBV）的除人类预大鹏编码基因的数量。这些可以被用来作为积极或消极的控制，取决于细胞株和所使用的样本的病毒状态。在图2中，一式四份运行KSHV的阴性样品平均的CT所示。重要的是，KSHV的LANA没有检测到在此示例中高度敏感的qPCR阵列。然而，U6 - 我国企业内部控制和let - 7A - 一个高度表达的人类前小分子RNA，表示检测不到的水平。最后，正如所料，PCR级水阴性对照没有产生的qPCR产品。 一个成功的qPCR运行的另一个指标是一个很好的的熔解曲线分析，可以洞察到样品中的潜在污染。要建立一个熔化温度，缓慢加热后的PCR完成板。由于DNA链分开，SYBR GREEN被释放，并降低荧光信号。温度在这个博士OP发生作图，作为样品的熔融温度。根据DNA序列，样品融化在彼此和水阴性对照，不应该有一个熔化温度，因为没有DNA存在不同的温度。从每对引物的PCR产物，应该在类似的温度融化。例如，图3显示了两个不同的引物重复使用同一样品的熔解曲线分析。很明显的熔化温度不同，但两个引物对样本是在完全相同的温度融化为每个复制。坏或可能受到污染的样品的迹象，可能包括几个熔融峰，表明存在两个不同的输入源。 <img src="/files/ftp_upload/2210/2210fig1.jpg" alt="图1" /> 出现图1。前的microRNA签名通过使用一种新型的qPCR基于阵列分析。平均deltaCTU6计算ArrayMinerTM软件装入标准值，高产heatmaps显示3个不同的集群。 （一）前的microRNA表达的小变化显示。前小分子RNA，其水平显着下降（B）或增加（三）也显示。蓝对应的表达水平较低，而红色代表表达水平增加。 <img src="/files/ftp_upload/2210/2210fig2.jpg" alt="图2" /> 图2:在qPCR的基于前的microRNA阵列的内部控制的共享。 3种不​​同的基因和无模板控制的原始CT值显示。 U6是用来作为内部阳性对照，PCR检测水作为我们的阴性对照服务。让- 7A - 1 - 1是通常在许多不同类型的细胞表达的microRNA，可以作为一个积极或消极的控制基于细胞系或组织使用的病毒状态，而KSHV的LANA。 <img src="/files/ftp_upload/2210/2210fig3.jpg" alt="图3" /> 图3。熔融峰前的小分子RNA引物和缺乏样品污染的分析。在LightCycler软件的使用TM调用分析熔解曲线获得。重复运行同一样品的两个不同的引物（底漆和B）所示。每个引物的熔融温度是不同的。然而，该样本只有一个高峰，展示了样品污染缺乏。

<ol>
	<li>Bustin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+Guidelines:+Minimum+Information+for+Publication+of+Quantitative+Real-Time+PCR+Experiments.">The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.</a> Clinical Chem. 55 (4), 611-622 (2009).</li><li>Fr&#233;geau, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Processing+of+Forensic+Casework+Samples+using+Robotic+Workstations+Equipped+with+Nondisposable+Tips+-+Contamination+Prevention.">Automated Processing of Forensic Casework Samples using Robotic Workstations Equipped with Nondisposable Tips - Contamination Prevention.</a> J. Forensic Sci. 53 (3), 53-533 (2008).</li><li>O'hara, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-micro+RNA+Signatures+Delineate+Stages+of+Endothelial+Cell+Transformation+in+Kaposi+Sarcoma.">Pre-micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma.</a> PLoS Pathog. 5 (4), e1000389-e1000389 (2009).</li></ol>

QPCR是一个高度敏感的检测可以用来比较样本之间的基因表达水平，在一项研究中，以及在病毒的情况下，以确定阳性。使用的qPCR阵列的好处是每个样品许多引物（在我们的例子中，186对引物），在很短的时间内运行的能力。使用特餐自由EVO，设置可以进一步减少实验所需的时间，和机器人的准确性和一致性，如移液机器人，可以减少和消除移液错误。在这里，我们将演示如何使用特餐自由埃沃设立的microRNA阵列，以及作为一种替代方法，使用矩阵电子多道移液器，谁没有访问一个移液机器人实验室，可用于实施。的qPCR阵列也是有利的，因为所有的基因，可以归到一个单一的同一个盘子上运行的参考基因，降低成本和反应。 正如前面提到的，有几个主要特点，设计这样的一个数组时所必需的。由于PCR板所有样品将在相同的条件下运行，这是设计引物，在相同的温度和设置，或一些对引物函数将失败的关键。设计引物时，应始终被检查的序列BLAST搜索，以确保有您的利益，没有其他基因的基因同源性。 当运行的阵列，它是关键，环境是无菌的，PCR的污染物，可能会导致误报。如果可能的话，PCR应设立在无菌工作橱或一个独立的洁净室，PCR产物是从来没有使用过。应洗净，用长凳Eliminase，水和乙醇，而遭受至少一个小时的紫外线照射后，运行完成。 这项技术的好处是，同样的策略是适用于任何设置，只需更换一个数组包含一个不同的引物对集的miRNA阵列异形基因。我们的实验室目前包含前的miRNA，KSHV的，EBV，P53，和NFKB阵列 - 所有这一切都可以在相同的方法所显示的运行。这个实验的灵活性，使我们的实验室快速，可靠地执行，我们希望屏幕的任何引物对高通量基因分析。 在这里，我们描述了在激活后，使用人类细胞系的cDNA阵列分析miRNA表达水平超过一段时间。此外表征细胞株中，数组也可以被用来分析临床标本。 RNA分离，cDNA合成，并可以运行使用这个数组来描述基因表达的cDNA。这可以有助于确定病毒阳性，或基因的表达谱，以进一步确定哪些基因是从受感染患者的样本上调。总之，自动定量PCR基于阵列，使高度敏感的，各种各样的及时组织和细胞株的基因检测的重复性和可靠的检测。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Freedom Evo 150</td>
<td>&nbsp;</td>
<td>Tecan</td>
<td>30017587</td>
<td>&nbsp;</td>
<tr>
<td>Matrix Electronic Multichannel Pipette</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>2001-MTX</td>
<td>(or any comparable Thermo Scientific multichannel that can pipette the volumes described above)</td>
</tr>
<tr>
<td>Matrix Integrity Filter Tips, Sterile</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7435</td>
<td>(or comparable tip for multichannel used)</td>
</tr>
<tr>
<td>Lightcycler 480 SYBR Green 1 Master</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>04 707 516 001</td>
<td>&nbsp;</td>
<tr>
<td>Lightcycler 480 Instrument II</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05015243001</td>
<td>384-well version</td>
</tr>
<tr>
<td>Lightcycler 480 Multiwell Plate 384, white</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>04729749001</td>
<td>&nbsp;</td>
<tr>
<td>LightCycler 480 Sealing Foil</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>04729757001</td>
<td>&nbsp;</td>
<tr>
<td>LightCycler 480 Multiple Plate Analysis Software</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05075122001</td>
<td>&nbsp;</td>
<tr>
<td>Eliminase Decontaminant</td>
<td>&nbsp;</td>
<td>Decon Laboratories</td>
<td>04-355-31</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

profiling of pre-micro rnas and micrornas using quantitative real-time pcr (qpcr) arrays

实时定量PCR（QPCR）已成为一个准确和有价值的工具，在分析基因表达水平。它的诸多优点之一，是一个较低的检测限相比，基因表达分析等方法，同时使用，每个检测数额较小的输入。自动定量PCR安装更大的可重复性，从而改善了这一领域。它的方便和快速设置允许用于高通量实验，使许多不同的基因分析，同时在每个实验。这连同内部板控制的方法，也降低了其他技术的共同实验变量。我们最近开发的一个前小分子RNA分析的定量PCR检测（前- miRNA的），使用186个引物对。小分子RNA已成为一类新的小，非编码RNA的调节能力，在许多基因的转录后水平的目标。作为一个主要的miRNA（PRI - miRNA）的成绩单，然后到前体miRNA（预miRNA）的切割，这些小分子RNA是由RNA聚合酶Ⅱ转录。预miRNA是出口到细胞质中Dicer酶切割产生成熟的miRNA发夹环。在miRNA水平的提高，可以观察到的前体和成熟的miRNA的水平都和这两种形式分析是有用的。有几种市售成熟的miRNA的检测，但其成本高，可能会阻止从这个分析技术的研究人员。在这里，我们讨论了一个具有成本效益，可靠的分析前的miRNA，基于SYBR的qPCR的方法。 miRNA的前水平的变化往往反映了成熟miRNA的变化和成熟的miRNA的表达可以是一个有用的指标。然而，同时分析前的miRNA和成熟的miRNA可能是最佳的，因为他们可以作出贡献的非冗余信息，并提供处理成小分子RNA的见解。此外，这里描述的技术可以扩展，包括具体途​​径或病原体的其他库集的分析。

Watch this Scientific Journal Video about Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays at JoVE.com

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

我们将演示前的小分子RNA的设置和分析QPCR 96孔阵列以及使用机器人手用Thermo Scientific的矩阵多道移液器。

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

profiling-pre-micro-rnas-micrornas-using-quantitative-real-time-pcr

Nanyang Technological University

Research

JoVE Journal

Biology

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

17.2K 次观看.  University of North Carolina at Chapel Hill. 我们将演示前的小分子RNA的设置和分析QPCR 96孔阵列以及使用机器人手用Thermo Scientific的矩阵多道移液器。

视频: Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

科研

生物学

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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 ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...