我们承认卢尔德三角洲Arrueta安特克拉（昆虫学系，美国俄亥俄州立大学/ OARDC，伍斯特，俄亥俄州）提供的实验设置的帮助。我们感谢发送EAB幼虫样品贷瑞莎波兰博士（美国农业部森林服务，网络实名制）。帮助路易斯一个卡纳斯和Nuris中号阿科斯塔博士用显微镜设置赞赏。为这个项目提供了资金由国家拨俄亥俄州农业研究和发展中心，美国俄亥俄州立大学和联邦基金。

完整的协议与不同的步骤是描绘在图1的流程图。构成夹层的各个步骤，第一链cDNA合成，RNA提取和定量RT - PCR详述如下。 一，幼虫解剖<ol><li>在此过程的开始，地方白蜡窄吉丁，或EAB，幼虫在潮湿的纸巾，直到解剖执行。保持新鲜配制的1X的磷酸盐缓冲液在冰，或PBS，以保持整个解剖缓冲区冷。 </li><li>首先，修复的罚款清扫引脚的夹层板的EAB幼虫。然后，添加冷1X PBS夹层板。 </li><li>首先，杀头的清扫剪刀的罚款对幼虫。随后取出幼虫的最后腹节。 </li><li>钟表匠对使用产钳，电梯从幼虫尸体的角质层。然后，沿切口用一把剪刀的尸体的长度。采取切口，小心不要肠道破裂。 </li><li>小心地隔离其他组织如脂肪组织和结缔组织中肠组织。然后，在新鲜的1X PBS缓冲液冲洗组织，以确保没有fatbody污染。孤立肠转移到500μL预冷Trizol试剂1.5 mL Eppendorf管。 </li><li>接下来，隔离脂肪的身体组织，使用镊子，并转移到一个1.5毫升的Eppendorf管含500μL冷冻Trizol试剂。 </li><li>最后，从幼虫尸体隔离杀任何坚持组织照顾，不损伤组织的完整性，角质层组织。在新鲜的PBS缓冲液冲洗隔离角质层组织和500μL冷冻Trizol试剂转移到1.5 mL Eppendorf管。 </li><li>隔离的脂肪体，角质层和肠组织可以储存在-80 ° C直到RNA提取。 </li><li>在RNA的提取准备，各种EAB样本进行排序，根据发展阶段。这些阶段包括第一，第二，第三，第四，龄期，prepupae和成人。 </li></ol>二。 RNA的提取（每制造商为使用Trizol试剂协议） 同质化<ol><li>要开始RNA的提取，首先使用塑料杵均质Trizol试剂含有1.5 mL的Eppendorf管组织。同质化后，带来Trizol试剂每个样品TO1毫升的总量。 </li><li>对于处于发育阶段，同质化在液氮用迫击炮和杵整个动物。然后加入1毫升的Trizol匀浆等分（50微克）。 </li></ol>相分离行为： <ol start="3"><li>用1 mL Trizol试剂在室温下15分钟的孵育样品管。 </li><li>孵化后，每管200μL氯仿。立即加入氯仿后，大力摇管约15秒。然后，在室温下孵育管额外2-3分钟。 </li><li>接下来，离心15分钟的样品在2 ° C，12,000 XG离心后，将在水相中的RNA。水相的体积应该是600μL，或60％的总Trizol法体积。 </li></ol> RNA沉淀<ol start="6"><li>小心地取出只有水相。以下的水相物质的存在会导致提取的RNA污染。然后，水相转移到一个适当的标记的1.5 mL Eppendorf管。 </li><li>从水相中的RNA沉淀，混合每管0.5毫升的异丙醇。样品在室温下孵育10分钟。孵化后10分钟，离心管，在2 ° C 12000 XG </li></ol> RNA的洗<ol start="8"><li>离心后，弃管的上清液。 RNA是在试管底部形成的凝胶体状的颗粒。用75％乙醇洗涤沉淀，增加至少1毫升75％，每1毫升的Trizol乙醇。混合振荡。然后5分钟，离心管，在2 ° C 7500Xg </li></ol> RNA的洗脱<ol start="9"><li>丢弃上清液管。再次在1分钟的小桌子离心机离心管。从试管中取出，小心吹打任何多余的上清液。离开管打开，让沉淀空气干燥5-10分钟。要小心不要过分干燥的颗粒，因为这将大大降低其溶解度。 </li><li>空气干燥后，重新悬浮颗粒<a href="http://en.wikipedia.org/wiki/Diethylpyrocarbonate">酸二乙酯</a> ，或DEPC处理，处理后的水。水用于悬浮颗粒的数量将取决于颗粒的大小。使用50μLDEPC处理水为SM全部为大颗粒的沉淀或100μL。让沉淀完全DEPC处理水溶解移动枪头向上和向下几次。 </li><li>一旦沉淀溶解，放置在55 ° C，10分钟的样品。 </li><li>然后，测量每个RNA样品的浓度，使用Nanodrop分光光度计（2μLRNA样品）。 </li><li>的RNA样品存放在-80 ° C，直到进一步的使用。 </li></ol>三。第一链cDNA合成（由于每个制造商使用标第一链合成试剂盒由Invitrogen S协议。） <ol><li>对于每个RNA样品在PCR管中添加以下内容： <table border="0"><tbody><tr><td>核糖核酸</td><td> xμL </td></tr><tr><td> 10MM的dNTP组合中号</td><td>加入1μl </td></tr><tr><td>以oligo（dT）（0.5μg/μL的） </td><td> 1μL </td></tr><tr><td> DEPC处理水</td><td> （8 - X）μL </td></tr><tr><td> 总成交量： </td><td> 10μL </td></tr><tr><td colspan="2">注：X是用于cDNA合成的RNA的量。 2-3微克的RNA取决于RNA的浓度，将用于每一个反应，每个反应总体积应该是10μL。 </td></tr></tbody></table></li><li>将上述反应混合在热循环在65 ° C，4分钟。 </li><li>从theromocyler取出的管子和至少2分钟，立即放在冰上。 </li><li>第一链预混准备加入以下： <table border="0"><tbody><tr><td> 10X RT缓冲</td><td> 2μL </td></tr><tr><td> 25MM氯化镁2 </td><td> 4μL </td></tr><tr><td> 0.1M的数码地面电视</td><td> 2μL </td></tr><tr><td>核糖核酸酶进行</td><td> 1μL </td></tr></tbody></table></li><li> 9预混液添加到每个试管中的RNA样品，由pipeting混合和离心机的混合物简要。 </li><li>管放置在热循环和42 ° C孵育2分钟</li><li>暂停的热循环，并添加1μL标II逆转录酶（Invitrogen公司），以每管。这一步应该是迅速完成。 </li><li>样品在42℃孵育1.5小时。 </li><li>终止反应，在70℃15分钟。 </li><li>删除从热循环管，并置于冰上。 </li><li>每个试管中加入1μl的RNase H的，将它们放置在热循环在37℃20分钟彗星。 </li><li>热循环仪的样本后，在每管加入20μL无核酸酶水。每个样品量的一倍，在这个阶段。 </li><li>检查使用2μL的样品，每个样品的浓度。每个样品等分，使用QRT - PCR20ng/μl的浓度。适量的水，以弥补样品浓度的20ng/μl将取决于初始浓度的合成cDNA。 </li></ol>四。 QRT - PCR： 参照基因的引物设计和测定<ol><li>与熔融温度为60℃，产品尺寸〜100bp（TM）设计引物。 </li><li> AP - PERI1是用来作为本实验的基因利益。一个参考基因或内部控制是需要的数据以供日后分析和正常化。核糖体蛋白（AP - RP1）作为本实验的参考基因。 </li><li>准备将用于引物，包括参照基因5um的股票。 </li></ol>制备标准<ol start="4"><li>为了计算标准应被用来引物的效率。 </li><li>制作的cDNA混合从不同的样本，将测试。 </li><li>图中的每个点5倍的系列稀释。四稀释应该足够，但五是高度推荐。 </li><li>多少分多少技术复制正在使用量会有所不同而有所不同。应该有足够使包括参照基因测试每一个基因的标准。 </li></ol>板模板<ol start="8"><li>设计模板QRT - PCR板，每孔样品名称。请记住，包括每个基因的标准，至少有两个技术是每个样本重复。 </li></ol>设立循环参数<ol start="9"><li> QRT - PCR的机器上打开和进入循环参数。这个实验的循环参数如下： 第1步： <ul><li> 1个循环：95℃3分钟</li></ul> 第2步： <ul><li> 40个循环：95 ° C 15秒60℃30秒，其次是 （可选：包括以下步骤确定熔解曲线） </li></uL&gt; 第3步： <ul><li> 1个循环：95℃1分钟</li></ul> 第4步： <ul><li> 1个周期：55℃1分钟</li></ul> 第5步： <ul><li> 81个循环：55℃30秒</li></ul></li></ol> CFX96（BioRad公司）机板成立<ol start="11"><li>准备掌握组合中的每个单独的管（包括参照基因）的基因。每口井，由主混音<table border="0"><tbody><tr><td>核酸免费水</td><td> 2μL </td></tr><tr><td> 2.5 × SYBR GREEN </td><td> 4μL </td></tr><tr><td>底漆向前</td><td> 1μL </td></tr><tr><td>底漆反向</td><td> 1μL </td></tr><tr><td> 总成交量 </td><td> 8μL </td></tr></tbody></table></li><li>虽然准备的主结构包括1-2帐户额外的移液错误反应。 </li><li>根据先前设计的模板，添加2μL（20毫微克/μL）的cDNA，每孔。然后，添加8μL的主结构，每孔，导致总量的10％μL反应。 </li><li>移液器向上和向下（2 - 3X），以确保样品充分混合。检查没有液体停留在刀尖。为了避免交叉污染，每个样品使用一个新的提示。 </li><li>一旦整个板块的设置，覆盖板，光学磁带。避免触摸磁带作为油脂的存在可以影响阅读。然后，离心1分钟的转速在500板，以确保所有产品在井是在盘底。 </li><li>离心后，检查有没有冰或水盘底。最后，放置在QRT - PCR机板和运行PCR程序。 </li></ol>五实验图解<img src="/files/ftp_upload/1974/1974fig1.jpg" alt="图1" /> 图1：流程图描绘的基因表达研究中的步骤的顺序。 <img src="/files/ftp_upload/1974/1974fig2.jpg" alt="图2" /> 图2：一）幼虫的EAB在中间夹层显示肠。二）孤立肠幼虫的EAB <img src="/files/ftp_upload/1974/1974fig3.jpg" alt="图3" /> 图3：a）平均为peritrophin基因在不同的幼虫组织，包括角质层（铜），肠（MG）和脂肪体（FB），（AP - PERI1）的相对表达值（ REVS）。 EAB特定的核糖体蛋白（以下简称命名，AP - RP1）的正常化感兴趣的基因，AP - PERI1获得的数据作为内部控制。 b）相对倍数变化AP - PERI1幼虫组织。角质层组织表明，至少表达，因此，校准器（1X）样本（Pfaffl 2001年）。 <img src="/files/ftp_upload/1974/1974fig4.jpg" alt="图4" /> 图4：a）平均为peritrophin基因在不同发育阶段的EAB包括幼虫（第一，第二，第三和第四），prepupae（PP）和成年人（一）（AP - PERI1）的相对表达值（REVS） 。 EAB特定的核糖体蛋白（AP - RP1）的正常化感兴趣的基因，AP - PERI1获得的数据作为内部控制。 b）相对在不同发育阶段的变化对AP - PERI1倍。 PP抽样调查表明mRNA水平的水平，因此，校准器（1X样品）。 六。结论实时定量PCR（QRT - PCR）是一种有效的工具来诊断，在不同的昆虫组织和发育阶段的mRNA水平。此外，QRT - PCR大多是从高通量基因表达分析，如芯片和新一代的RNA - Seq的产生的数据的主要工具，以验证。

<ol>
	<li>Lehane, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritrophic+matrix+structure+and+function.">Peritrophic matrix structure and function.</a> Annu Rev Entomol. 42, 525-550 (1997).</li><li>Mittapalli, O., Sardesai, N., Shukle, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cDNA+cloning+and+transcriptional+expression+of+a+peritrophin-like+gene+in+the+Hessian+fly,+Mayetiola+destructor+[Say].">cDNA cloning and transcriptional expression of a peritrophin-like gene in the Hessian fly, Mayetiola destructor [Say].</a> Arc of Insect Bio. 64, 19-29 (2007).</li><li>Pauchet, Y., Wilkinson, P., van Munster, M., Augustin, S., Pauron, D., ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing+of+the+midgut+transcriptome+of+the+poplar+leaf+beetle+Chrysomela+tremulae+reveals+new+gene+families+in+Coleoptera.+Ins+Bio+and+Mol.">Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera. Ins Bio and Mol.</a> 39, 403-413 (2009).</li><li>Pfaffl, W. 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+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29, 2002-2007 (2001).</li><li>Tellam, R. L., Lehane, M. J., Billingsley, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of the Insect Midgut. , 86-114 (1996).</li></ol>

获得每个样品定量RT - PCR板的阈值循环（CT）的价值。为了使标准曲线，Ct值，每个稀释度获得暗算其浓度的日志。实验样品的CT值，然后到这个系列稀释标准曲线的绘制。目标数量分别计算每一个实验，即组织和发育中的表达产生不同的标准曲线。相对表达值（REVS），然后确定内部控制获得的数量（AP - RP1）除以利益的目标序列的数量（在这种情况下，AP - PERI1 ）。计算的意义，每个...

analysis of gene expression in emerald ash borer (agrilus planipennis) using quantitative real time-pcr

白蜡窄吉丁（EAB，<em&gt; Agrilus planipennis</em&gt;）是一种外来入侵害虫，已造成以百万计的白蜡树（<em&gt;白腊</em&gt; SPP）在北美。<strong&gt;</strong&gt; EAB继续迅速传播和攻击灰不同年龄的树木，从成熟的树木树苗。然而，迄今为止很少或没有分子知识的EAB存在。我们感兴趣的是破译的分子为基础的生理过程，在组织水​​平，援助的EAB成功定植白蜡树。在这份报告中，我们有效地利用实时定量PCR（QRT - PCR），以确定在不同的幼虫组织（包括肠，脂肪体和角质层）和不同发育阶段（包括1的表达水平<sup&gt; ST</sup- 2<sup&gt; ND</sup&gt; - ，3<sup&gt; RD</sup- 4<sup&gt; TH</sup&gt;龄，prepupae和成人）的EAB。作为一个例子，一个peritrophin基因（本文命名，<em&gt; AP - PERI1</em&gt;）例证感兴趣的基因和核糖体蛋白（<em&gt; AP - RP1</em&gt;）作为内部控制。 Peritrophins围食膜/矩阵（PM），这是昆虫肠道内壁的重要组成部分。下午有多种功能，包括消化和机械保护中肠上皮细胞。

Watch this Scientific Journal Video about Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR at JoVE.com

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

实时定量PCR（QRT - PCR）是一种有效的工具来诊断，在不同的昆虫组织和发育阶段的mRNA水平。在这份报告中，我们使用QRT - PCR在不同的幼虫组织确定的mRNA水平和侵入性的昆虫物种，白蜡窄吉丁的发展阶段。

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 ...

analysis-gene-expression-emerald-ash-borer-agrilus-planipennis-using

Research

JoVE Journal

Biology

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

10.6K 次观看.  The Ohio State University. 实时定量PCR（QRT - PCR）是一种有效的工具来诊断，在不同的昆虫组织和发育阶段的mRNA水平。在这份报告中，我们使用QRT - PCR在不同的幼虫组织确定的mRNA水平和侵入性的昆虫物种，白蜡窄吉丁的发展阶段。

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

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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 ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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, ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...