Name: processing the loblolly pine ptgen2 cdna microarray
Uploaded: 2009-03-20T00:00:00
Description: Watch this Scientific Journal Video about Processing the Loblolly Pine PtGen2 cDNA Microarray at JoVE.com

的标签，杂交，洗涤协议本文包含一些修改，而在基因组研究所（TIGR），肖恩 - 利维博士范德比尔特芯片共享资源（VMSR）研究所博士J. Quackenbush以前开发的博士和罗布阿尔巴而博伊斯汤普森研究所（BTI）的康奈尔大学。

（注意：1 - 4节不在视频展示） 准备CY -标记的目标基因下面是我们使用cDNA合成火炬松总RNA和间接的基因标签芯片杂交前的协议。虽然已作出一些非平凡的修改，下面的协议是与Invitrogen公司的间接标基因标记试剂盒提供的说明书中的类似。 第1部分：第一链cDNA合成反应<ol><li>每个套件的组件在使用前混合，并简要旋转。 </li><li>准备反...

<ol>
	<li>Lorenz, W. W., Sun, F., Liang, C., Kolychev, D., Wang, H., Zhao, X., Cordonnier-Pratt, M. M., Pratt, L. H., Dean, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+stress-responsive+genes+in+loblolly+pine+(Pinus+taeda)+roots+identified+by+analyses+of+expressed+sequence+tag+libraries.">Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries.</a> Tree Physiol. 26, 1-16 (2006).</li><li>Lorenz, W. W., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> unpublished data. , .</li><li>Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E., Snesrud, E., Lee, N., Quackenbush, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concise+guide+to+cDNA+microarray+analysis.">A concise guide to cDNA microarray analysis.</a> Biotechniques. 29, 548-562 (2000).</li><li>Alba, R., Fei, Z., Payton, P., Liu, Y., Moore, S. L., Debbie, P., Cohn, J., D'Ascenzo, M., Gordon, J. S., Rose, J. K. C., Martin, G., Tanksley, S. D., Bouzayen, M., Molly, M., Jahn, M. M., Giovannoni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESTs,+cDNA+microarrays,+and+gene+expression+profiling:+tools+for+dissecting+plant+physiology+and+development.">ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.</a> The Plant Journal. 39, 697-714 (2004).</li><li>Lorenz, W. W., Simões, M., Miguel, C., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Gene+Expression+changes+in+Pinus+species+using+a+Loblolly+pine+cDNA+microarray.">Analysis of Gene Expression changes in Pinus species using a Loblolly pine cDNA microarray.</a> IUFRO-CTIA 2008 Joint Conference. , (2008).</li><li>Simões, M., Lorenz, W. W., Alba, A., Gonçalves, S., Dean, J., Miguel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+during+P.+pinaster+embryo+development.">Global gene expression analysis during P. pinaster embryo development.</a> 7th Plant Genomics European Meeting. , (2008).</li></ol>

PtGen2是最近发展起来的，定制的cDNA微阵列技术，主要是设计用来火炬松研究社会。迄今产生的初步结果表明数组是一个有价值的工具，在转录事件，在干旱胁迫反应的发生，以及在监测胚胎发育过程中发生的变化，在海上松 ，马尾松松树5,6我们的评价最近还证明PtGen2工程，以及在跨物种杂交利用多元化的针叶树属孤立的目标样本。变得越来越松和其他针叶树种在研究基因?...

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<td>Pre-hybridization Buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
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<td>Hybridization Buffer</td>
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<td>30% formamide, 5X SSC, 5X Denhardt&#x2019;s, 1% PolyA RNA (Invitrogen, POLYA.GF), 0.1% SDS</td>
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<td>Wash Solution #1</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X SSC, 0.2% SDS, preheat to 43&deg;C</td>
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<td>Wash Solution #2</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>34959-2.5L</td>
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<td>Coplin Jar</td>
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<td>&nbsp;</td>
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<td>Cy-5 Dye</td>
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<td>HybChambers</td>
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<td>GeneMachines</td>
<td>JHYB200003</td>
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processing the loblolly pine ptgen2 cdna microarray

PtGen2是一个26496的功能包含放大的火炬松松树EST序列的cDNA微阵列。松树和其他针叶树种基因的表达研究的研究人员所使用的阵列是在我们的实验室生产。 PtGen2发展作为我们的基因中发现火炬松努力的结果，并主要从根组织确定的序列组成，但也从针和干1,2 PtGen2已通过杂交不同的针叶树目标CY -染料标记的cDNA测试使用放大和非扩增间接标记的方法，也与杂交和洗涤条件进行测试。此影片的重点预杂交前后幻灯片的处理和加工，以及杂交后，用一些发达国家以前的程序进行修改。3,4还包括，只以文本形式，是为一代人所使用的协议，用于下行数据处理标签和目标的cDNA小号清理，以及对软件的信息。 PtGen2是印有一个专有的打印缓冲区，含有高浓度的盐，可很难完全消除。第一次在一个温暖的SDS溶液前预杂交幻灯片冲洗。预杂交后，幻灯片冲洗水的几个变化，积极完成拆除剩余的盐。 LifterSlips™然后清洗和幻灯片上的定位标记的cDNA仔细加载到芯片由毛细作用的方式，甚至整个幻灯片的样本分布，并减少泡沫法团的机会。杂交阵列目标是在48℃，在高湿度条件下完成。杂交后，一个标准洗涤系列53 ° C和室温延长时间。加工PtGen2使用这种技术的幻灯片，减少盐和经常看到的SDS -派生文物数组时不太严格处理。杂交从几个不同的针叶树RNA的来源派生的目标，这种处理协议取得更少的文物，减少背景，和不同的实验组阵列之间提供更好的一致性。

Watch this Scientific Journal Video about Processing the Loblolly Pine PtGen2 cDNA Microarray at JoVE.com

Processing the Loblolly Pine PtGen2 cDNA Microarray

的cDNA微阵列PtGen2的基因表达研究开发火炬松，<em&gt; P。火炬松</em&gt;，和其他针叶树种。在这里，我们展示了前和杂交后处理和清洗技术，可以使用这个数组产生更好的一致性，减少了工件，和较低的背景。

处理火炬松PtGen2差异表达基因的cDNA微阵列

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

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