Name: analysis of gene expression in emerald ash borer (agrilus planipennis) using quantitative real time-pcr
Uploaded: 2010-05-04T17:00:00
Description: Watch this Scientific Journal Video about Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR at JoVE.com

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。...

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	<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 ...

Research

JoVE Journal

Biology

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

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

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 ...

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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

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

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

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

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

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

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

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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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

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可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...