我们衷心感谢T. Ragucci，B.沃特金斯，S.黄，二Wallek Lynntech，公司提供技术援助和许多有益的讨论。这项工作是由美国国家科学基金会（NSF）资助CBET - 0933688的支持。

1。对流热循环的基本设计<ol><li>我们已经构建了一个简单的对流热循环设备可互换的塑料反应室“墨盒”是两个铝板的温度可以独立控制（图2）之间夹着组成。 </li><li>圆柱反应堆井嵌入式加工聚碳酸酯块阵列孔与孔的直径和塑料板的厚度，以达到所需的高宽比（高度/直径= H / D）的不同组合。在这里提出的结果被认为是两种不同的反应器几何：H / D = 9：H = 15.9毫米，D = 1.75毫米，38.2μL量; H / D = 3：H = 6.02毫米，D = 1.98毫米，18.5微升音量。 </li><li>该反应器的底部表面被加热使用铝块，其中包含墨盒加热器与microprocesser驱动温度控制器接口。 </li><li>在反应器的上表面的温度调节使用铝块连接到一个循环水浴。 </li><li>整个大会钳制在一起使用尼龙螺丝限制反对的铝块之间的热传导。 </li></ol> 2。 PCR反应的制备和载入<ol><li>一个典型的100μL反应混合物中含有MgCl 2的 10μL10X缓冲液中，25毫米6μL，10μL，dNTPs（各2毫米），41.2μL去离子水，10μLβ-肌动蛋白探针，10μLβ肌动蛋白正向引物，10μLβ-肌动蛋白的反向引物，2μL的人类基因组DNA模板（10毫微克/μL）和KOD ​​DNA聚合酶（2.5个/μL）0.8μL。 </li><li>在装载试剂，密封的PCR室底部的表面，使用铝带的薄板。 </li><li>与10毫克/毫升的水，牛血清白蛋白有雨- X的防雾的解决方案冲洗反应堆井。 </li><li>进入反应堆井用长胶装枪头移液器试剂和铁氟龙胶带密封上表面。 </li></ol> 3。运行中的对流热循环反应<ol><li>在开始反应，预热铝块所需的上部和下部的表面温度。 </li><li>夹心塑料反应堆装有铝加热块之间的PCR试剂，并迅速用尼龙螺丝钳大会。 </li><li>所需的时间进行反应后，关掉加热器和地方的低（热）设备表面冷冻的金属块，迅速冷却，并停止对流顶部。 </li><li>取下塑料反应堆和吸管井（用长胶加载提示）的产品，以供后续分析。 </li></ol> 4。执行凝胶电泳分析<ol><li>准备2％琼脂糖凝胶加热一种震撼人心的热点板块1X缓冲液500毫升10克的琼脂糖，直到解决方案变得清晰。 </li><li>装入托盘铸造的琼脂糖凝胶电泳，插入梳子。让凝胶集，约30分钟。 </li><li>取出梳子和1X TAE缓冲液中添加，直到被淹没凝胶。 </li><li>荧光染色的DNA样本包含2μL，100X SYBR GREEN我的解决方案，2μL的DNA样本，2μL6X橙色样染料，和4μLTAE缓冲液。 </li><li>添加入井的DNA样本，并运行在60 V与100 bp的DNA梯状条带大小的标记1小时的分离。 </li><li>取出凝胶和照片，在紫外光下获得的结果。 </li></ol> 5。代表性的成果： 对流热循环的优化设计，包括选择正确的反应器的几何形状，会产生一个循环流动，能够通过在PCR过程中所涉及的主要温度运送试剂。可以在这里考虑的圆柱形反应堆不同的几何参数的高度（H）和直径（d），或等价的高宽比（H / D） 。我们已经探索的3 - D流领域，通过一系列不同的方面使用计算流体力学（CFD）的比率PCR反应器内对流和发现可能会出现意外复杂的图案。更重要的是，我们的分析发现这些复杂的流场，大大加快反应 12的一个子集。 反应堆比较高，低长宽比的流场，可以清楚地看到这些几何效应。在高纵横比的情况下（H / D = 9;一个高个子，瘦的缸），是平流输送流体元素沿轨迹跟踪，基本上都是封闭的循环路径，他们锁定的长期遵循相同的路径（图3a）。因此，有交流的机会很少，在F，暴露试剂的PCR扩增的退火和变性反应器的顶部和底部表面极端之间振荡，并在余下的轨迹更大的合奏，做不作出贡献，以扩增（本地化更接近中心热条件最优序列低轨迹反应堆）。 在一个较小的长宽比（H / D = 3;更短，更广泛的缸）的流场有很大不同，变得更加混乱的意识，流体元素轨迹不能再走封闭路径（图3b）。因此，即使试剂受到更复杂的温度曲线，流体元素能够探索出一条轨迹的范围更广，使更多的试剂量有机会体验到最佳的温度曲线。这些结果直觉相反建议，个别流动轨迹，而在H / D = 9，可能会出现产生的温度曲线进行PCR更为有利的，类似于在一个传统的热循环，混沌性质的流场，在 H /就业的“看“ D = 3，最终在全球范围内占主导地位，促进加强交流，使试剂不会成为被困在不利的轨迹很长。 为了检验这一假设，并确定这更有利于进行PCR反应器几何，我们用他们两个执行一个295 bp的目标从人类基因组DNA为模板的β-肌动蛋白基因PCR复制。值得注意的是，我们经常达到正确的目标产品的放大，在只有10分钟在H / D = 3（图4a），而同样的反应，检测的产品之前，需要至少20 分钟，在 H / D = 9（图4b） 。反应的特异性也远在 H / D = 3获得一个单一的PCR产物，而多个非特异性产物生成在 H / D = 9，流场的陷阱流体元素在长时间的不利的热轨迹。 <img src="/files/ftp_upload/2366/2366fig1.jpg" alt="图1" /> 图1：在一个圆柱形腔的顶部和底部表面的热对流，保持固定在不同温度。如果在底部表面温度高于顶部，垂直密度梯度是建立在封闭液是能够产生一个循环流动模式。对流的流场的几何参数（ 高度 H和直径 d）正确的选择，可以利用驱动PCR热循环时，顶部和底部表面附近退火和变性温度分别为（重力作用垂直向下）manintained 。 <img src="/files/ftp_upload/2366/2366fig2.jpg" alt="图2" /> 图2（A）一个对流的关键组成部分PCR扩增thermocyler。反应堆盒由聚碳酸酯块包含一个圆柱商会阵列。夹块之间要连接到一个循环水浴和底板结合嵌入式加热器设计一个顶板。 （二）PCR试 ​​剂加载和反应器内墨盒密封，组装后的设备进行反应。工程完成后，可以很容易吸管procucts供后续分析。 <img src="/files/ftp_upload/2366/2366fig3.jpg" alt="图3" /> 图3。计算机模拟的3 - D对流领域圆柱形反应堆内产生的温度在53和96 ° C的顶部和底部表面施加，分别。结果显示长宽比（ 一 ）H / D = 9（38.2μL的反应器体积）和（B）H / D = 3（18.5μL的反应器体积）。由流体的三维流场的元素是一个代表性的轨迹显示在左沿路径的顶视图和侧视图预测是在合适的温度随时间变化的流体元素相应的情节。在H / D = 9的温度曲线出现接近传统的热循环，但在H / D = 3的乱流场是直觉相反PCR更为有利。 <img src="/files/ftp_upload/2366/2366fig4.jpg" alt="图4" /> 图4。DNA复制反应器的几何结构显示在图3（上下表面均保持在53和96℃，分别）获得的结果。 （ 一 ）PCR是大大加快了H / D = 3，明显可见的强大的产品后的反应时间只有10分钟（M：100 bp的阶梯，车道1-4：产品在4个不同的圆柱腔平行反应）。 （二</strONG&gt;）在H / D = 9，执行相同的反应需要至少20分钟前有形产品，和多个频段所需的产品除了在明显表明非特异性的靶DNA的复制（M：100 bp的阶梯，泳道1-2：在2个不同的圆柱腔的平行反应的产品）。

<ol>
	<li>Spitzack, K. D., Ugaz, V. M., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Techniques:+Reviews+and+Protocols.">Microfluidic Techniques: Reviews and Protocols.</a> Methods in Molecular Biology. , (2005).</li><li>Krishnan, M., Ugaz, V. M., Burns, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+in+a+Rayleigh-B&#233;nard+convection+cell.">PCR in a Rayleigh-B&#233;nard convection cell.</a> Science. 298, 793-793 (2002).</li><li>Braun, D., Goddard, N. L., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exponential+DNA+replication+by+laminar+convection.">Exponential DNA replication by laminar convection.</a> Physical Review Letters. 91, 158103-158103 (2003).</li><li>Chen, Z., Qian, S., Abrams, W. R., Malamud, D., Bau, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermosiphon-based+PCR+reactor:+experiment+and+modeling.">Thermosiphon-based PCR reactor: experiment and modeling.</a> Analytical Chemistry. 76, 3707-3715 (2004).</li><li>Wheeler, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convectively+driven+polymerase+chain+reaction+thermal+cycler.">Convectively driven polymerase chain reaction thermal cycler.</a> Analytical Chemistry. 76, 4011-4016 (2004).</li><li>Krishnan, M., Agrawal, N., Burns, M. A., Ugaz, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+and+fluidics+in+miniaturized+natural+convection+systems.">Reactions and fluidics in miniaturized natural convection systems.</a> Analytical Chemistry. 76, 6254-6265 (2004).</li><li>Ugaz, V. M., Krishnan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+convective+flow+based+approaches+for+high-throughput+PCR+thermocycling.">Novel convective flow based approaches for high-throughput PCR thermocycling.</a> Journal of the Association for Laboratory Automation (JALA). 9, 318-323 (2004).</li><li>Yao, D. J., Chen, J. R., Ju, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-Rayleigh-Benard+convection+polymerase+chain+reaction+system.">Micro-Rayleigh-Benard convection polymerase chain reaction system.</a> Journal of Micro-Nanolithography MEMS and MOEMS. 6, 043007-043007 (2007).</li><li>Agrawal, N., Hassan, Y. A., Ugaz, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pocket-sized+Convective+PCR+Thermocycler.">A Pocket-sized Convective PCR Thermocycler.</a> Angewandte Chemie International Edition. 46, 4316-4319 (2007).</li><li>Yariv, E., Ben-Dov, G., Dorfman, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction+in+natural+convection+systems:+A+convection-diffusion-reaction+model.">Polymerase chain reaction in natural convection systems: A convection-diffusion-reaction model.</a> Europhysics Letters. 71, 1008-1014 (2005).</li><li>Allen, J. W., Kenward, M., Dorfman, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupled+flow+and+reaction+during+natural+convection+PCR.">Coupled flow and reaction during natural convection PCR.</a> Microfluidics and Nanofluidics. 6, 121-130 (2009).</li><li>Muddu, R., Hassan, Y. A., Ugaz, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+Biochemistry+by+Chaotic+Stirring.">Accelerated Biochemistry by Chaotic Stirring.</a> , (2010).</li></ol>

混乱平流的影响下，流动和化学反应之间的相互作用发生显着变化。但这些复杂的流动状态是具有挑战性的研究，特别是在逼真的3D效果成为重要的几何形状。瑞利 - 贝纳尔对流在H / D&gt; 1，不仅提供了一个方便的平台，探索这些现象，他们的应用程序进行PCR，也使被探测的流动和化学反应之间的耦合。这种独特的能力使我们能够选择最佳的流动状态混乱影响行为（直觉相反），以加速DNA的复制率。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>KOD DNA Polymerase kit</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>71085-3</td>
<td>&nbsp;</td>
<tr>
<td>TaqMan &beta;-actin Control Reagents kit</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>401846</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum tape</td>
<td>&nbsp;</td>
<td>Axygen, Inc.</td>
<td>PCR-AS-200</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2153</td>
<td>&nbsp;</td>
<tr>
<td>Rain-X Anti-Fog</td>
<td>&nbsp;</td>
<td>SOPUS Products</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Long Gel-loading Pipette Tips</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>05-408-151</td>
<td>&nbsp;</td>
<tr>
<td>FEP Teflon tape</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>7562A17</td>
<td>&nbsp;</td>
<tr>
<td>Agarose gel</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-3107</td>
<td>&nbsp;</td>
<tr>
<td>TAE running buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>141-0743</td>
<td>&nbsp;</td>
<tr>
<td>SYBR Green I</td>
<td>&nbsp;</td>
<td>Invitrogen/Molecular Probes</td>
<td>S7563</td>
<td>&nbsp;</td>
<tr>
<td>6x Orange Loading Dye</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>R0631</td>
<td>&nbsp;</td>
<tr>
<td>100 bp DNA ladder sizing marker</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8202</td>
<td>&nbsp;</td>		</tbody>
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rapid pcr thermocycling using microscale thermal convection

许多分子生物学实验取决于在一些聚合酶链反应（PCR）扩增最初稀的目标DNA样本检出浓度水平的方法。但常规的PCR热循环硬件的设计，主要是基于大量的金属加热块的温度是由热电热水器的监管，严重限制了可达到的反应速度1。大量的电力，还需要反复加热和冷却的试剂混合，限制部署在一台便携式的格式这些文书的能力。 已经成为一个有前途的替代热循环的方法，有可能克服这些局限性2-9的热对流。对流的流量每天都发生在一个多样化，包括从地球的大气层，海洋，内饰，装饰和丰富多彩的熔岩灯的设置的。流体运动开始以同样的方式，在每一种情况下：浮力驱动的不稳定发生时，密闭的液体量是受到空间的温度梯度。这些相同的现象提供一个有吸引力的的方式进行PCR热循环。应用跨一个适当设计的反应器几何的静态温度梯度，连续的循环流动，可以建立，将通过反复运输的变性，退火，和扩展阶段的反应（图1）温区的PCR试剂。在伪等温方式，热循环，因此可以被驱动，只需在固定温度下两种对立的表面，完全消除需要反复加热和冷却仪器。 对流热循环设计所面临的主要挑战之一是需要精确控制反应器内的空间的速度和温度分布，以确保试剂的顺序占据足够的时间10,11期的正确的温度区。在这里，我们描述了我们努力探索的全三维速度和温度分布在微尺度对流热循环12的结果。出乎意料的是，我们已经发现了复杂的流动轨迹进行PCR极为有利的，因为一个协同组合（1）流动路径之间的不断交流提供了一个增强的机会，试剂，样品的最佳温度廓线的全方位的一个子集，（ 2）增加花费的时间内延伸温度区的速率限制PCR步骤。极其迅速的DNA扩增时间（10分钟）在反应堆设计产生这些流动实现的。

Watch this Scientific Journal Video about Rapid PCR Thermocycling using Microscale Thermal Convection at JoVE.com

Rapid PCR Thermocycling using Microscale Thermal Convection

我们描述了一种新的方法，通过聚合酶链反应（PCR）进行DNA复制。热对流是利用变性，退火，以及延长条件之间不断穿梭试剂保持反对在反应器的表面温度恒定。这在本质上简单设计的承诺，使快速PCR更方便。

快速PCR热循环利用微型热对流

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

rapid-pcr-thermocycling-using-microscale-thermal-convection

Research

JoVE Journal

Biology

22.6K 次观看.  Texas A&M University. 我们描述了一种新的方法，通过聚合酶链反应（PCR）进行DNA复制。热对流是利用变性，退火，以及延长条件之间不断穿梭试剂保持反对在反应器的表面温度恒定。这在本质上简单设计的承诺，使快速PCR更方便。

视频: Rapid PCR Thermocycling using Microscale Thermal Convection

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

使用Sigma的提取物N -安培组织PCR试剂盒小鼠组织的快速基因分型

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

科研

生物学

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 ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

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 ...

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

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 detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

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

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

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 ...

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, ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

优化的基于PCR的检测支原体

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. 
This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

检测莱姆病螺旋体,螺旋体螺旋体, 在蜱中使用巢式 PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...