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>准备反应如下： <ul><li> XμlDEPC - H 2 O </li><li> Xμl总RNA，20微克/反应（Ambion公司的涡轮DNA酶处理） </li><li> 2μL锚以oligo（dT）20引物（2.5μg/μl ） </li><li>加入1μl的随机六聚体引物</li><li style="list-style:none;"> 总体积=18μl </li></ul></li><li>在70 ° C为5分钟，然后置于冰上1-2分钟孵育管。 </li><li>以下内容添加到每个反应管冰： <ul><li> 6μl5X第一链缓冲</li><li> 1.5μl0.1M数码地面电视</li><li> 1.5μl10MM dNTP混合液</li><li> RNaseOUT加入1μl（40U/μl） </li><li> 2μL标III RT（400U/μl） </li></ul></li><li>轻轻混匀，旋转简要。一夜之间管孵育45 ° C。 </li><li> 1M NaOH溶液15μl加入到每个反应管中，调匀。 </li><li>在70 ° C孵育10分钟管。 </li><li> 1M盐酸加15μl，轻轻混匀。 </li><li>加入20μL3M的醋酸钠（pH值5.2），轻轻混匀。 </li></ol>第2部分：净化第一链cDNA的。 <ol><li>载入缓冲区的cDNA（步骤1.9）中加入500μL和拌匀。 </li><li>一个收集管上放置一个SNAP纯化柱（间接标记试剂盒提供）和负载的列上你的基因。 </li><li>自旋14000克，在室温1分钟，丢弃流过。 </li><li> SNAP列和放置到同一个收集管加入洗涤缓冲液500μL。 </li><li>自旋14000克，在室温1分钟，丢弃流过。 </li><li>重复步骤2.4和2.5的三倍，共有三个500μL洗涤。 </li><li>在14000克旋转一个更多的时间在室温1分钟的秒;丢弃流过。 </li><li>放置到一个新的1.5毫升管的SNAP列。 </li><li>添加温暖DEPC水（50℃）50μL的SNAP列，并在孵化室温度为1分钟列。洗脱通过自旋为1分钟的房间温度在14,000克的cDNA。 </li><li>重复步骤2.9，使用温暖DEPC水100μL和相同的洗脱液1.5毫升管。 </li><li>添加20μL3M的醋酸钠（pH值5.2）步骤2.9和2.10的洗脱液。 </li><li>糖原（20mg/ml）3μl管和组合。 </li><li>冰冷的100％的乙醇添加500μL，拌匀，和管孵育1小时，在-80 ° C。 </li><li> 14000克旋转管在4 ° C 20分钟。小心去除上清。 </li><li>冰冷的70％的乙醇添加500μL和自旋14,000克管，在4 ° 5分钟的。小心去除上清。 </li><li>空气干燥5分钟的样品，确保所有被删除乙醇。 </li><li> 2X耦合缓冲液温在37 ° C 5分钟，重新暂停在温暖的2X耦合缓冲液5μLcDNA样本。 </li><li>的cDNA /耦合缓冲区的温度在50 °彗星以及10分钟和涡。确保您的cDNA颗粒在2X耦合缓冲区是完全重新悬浮。 </li></ol>第3部分：用荧光染料标签 准备反应时，要小心，尽量减少光的染料溶液的接触 。 此外，二甲基亚砜是吸湿性，会吸收空气中的水分，这将与染料NHS酯反应，并大大降低耦合反应效率 。 保持琥珀色的螺丝帽小瓶于-20 ° C试剂盒中提供的二甲基亚砜，让小瓶开放前，以防止结露温至室温。只能使用本试剂盒提供的二甲基亚砜 。 <ol><li>打开一包CY - 3或CY - 5染料和直接添加二甲基亚砜75μl染料小瓶。 </li><li>新增的二甲基亚砜/染料溶液5μL步骤2.18管。 </li><li>充分混匀，室温在黑暗中孵育1小时管。 </li><li>加入20μL3M的醋酸钠（pH值5.2）的染料耦合的cDNA解决方案。 </li><li>加入500μL样缓冲的cDNA的解决方案。由涡旋混合。 </li><li>放置到一个明确的收集管中的管理单元列和负载的cDNA /缓冲液。 </li><li>自旋14000克，在室温1分钟，丢弃流过。 </li><li>放在同一个集合的SNAP列管;洗涤缓冲液500μL添加到列。 </li><li>自旋14000克，在室温1分钟，丢弃流过。 </li><li>重复步骤3。 8-3.9 3次，共有三个500μL洗涤。 </li><li>在14000克旋转一个更多的时间在室温1分钟，丢弃流过。 </li><li>放置到一个新的琥珀收集管的SNAP列。 </li><li>添加温暖DEPC水（50℃）65μl的SNAP列，并在室温下孵育1分钟。 </li><li>旋转14000 克在室温1分钟，收集流过。流过，应该包含您的纯化染料耦合的cDNA60μl。 </li></ol>第4部分：评估的标记过程。 <ol><li>行为分光光度计的检测，以评估每个CY - cDNA的标签。计算合成cDNA pmol总数：外径260 ×卷。 x37ng/μl× 10 3毫微克/ PG] / 324.5 pg / pmol的。计算每个标记反应纳入CY -染料的总pmol：pmol Cy3标记= [ 外径550 X卷] / 0.15; pmol Cy5的= [OD 650 ×卷] / 0.25 。下一步，计算每个反应核苷酸/ pmol染料比率pmol。最优反应生成&gt; 6000pmol的基因（每60ul），CY - 200pmol染料（每60ul），核苷酸/染料比&lt;30。 </li></ol>第5部分：幻灯片预洗<ol><li> PtGen2阵列上印有康宁UltraGaps幻灯片（氨基硅烷涂层）和cDNA的紫外线交联。打印点都清晰可见，由于缓冲区的含盐量高。 </li><li>使用钻石尖端的笔，以纪念幻灯片的打印区域（可选）的上限和下限的地区。 </li><li>投入约250毫升预热到43 ° C 0.2％SDS到显微镜玻片洗菜（3“× 4”）的解决方案。 </li><li>广场幻灯片预杂交在幻灯片机架和投身到SDS溶液非常积极的15-20倍删除幻灯片上的盐。 </li><li>使用镊子，小心地从机架中删除的幻灯片，并放置在一个Coplin包含预杂交液（5X SSC，0.1％SDS，1％BSA）的JAR已预热至43 ° C在43 ° C孵育至少一小时。 </li></ol>第6部分：预杂交<ol><li>设置五个幻灯片洗碗，每次充满millipure DH 2 O，和一个Coplin JAR充满分子级异丙醇。广场的幻灯片，在幻灯片机架，五菜过程中，通过连续扣篮20次。 </li><li>删除幻灯片在两个时间从第五清水洗净，浸5-6倍，在幻灯片中的异丙醇，并立即到幻灯片离心（Chipmate台式，2个职位）和自旋为1分钟干。 </li><li>通过0.22微米滤芯过滤用压缩空气清洁幻灯片和他们面临的一个基因组的解决方案杂交商会与酒吧代码。 </li><li>清洁LifterSlips™过滤空气和朝下的白色条状幻灯片的位置。使用黄色吸管尖轻轻地对齐幻灯片上的升降机滑（70％的乙醇洗涤LifterSlips™首先是可选的。我们发现它是不必要的。） </li></ol>第7部分：杂交 应在黑暗或光线较暗的开展。 <ol><li>对于每一个将杂交幻灯片，计算金额的CY - 5和CY - 3标记的cDNA给每个目标的50 75pmol。合并成一个单管的金额在37-45在真空干燥器中干燥热定型° C。完全避免干燥！ </li><li>在60ml的杂交缓冲液重悬的干探头（新鲜那天）和涡简要。 </li><li>孵育在95℃的浴缸和30秒快速离心5分钟，悬浮的cDNA。 </li><li>载入缓慢幻灯片LifterSlip™交界处的底部的幻灯片（条形码完）吹打整个悬浮探头量。幻灯片将载入毛细作用的方式。 （有些人喜欢吸管上的幻灯片，然后轻轻地覆盖LifterSlip™这是一个一杆的方法，因为如果你得到气泡不能移动后的位置，从而导致现货涂抹文物LifterSlip™） </li><li> 100mm的数码地面电视将20uL到每个湿度以及位于香港总商会的两端。 </li><li>广场上腔的顶部，并拧紧凸轮螺丝。盖到48℃的水浴12-18小时，轻轻摇动，在50-60转用铝箔和沉浸室。 </li></ol>第8部分：杂交后洗 所有清洗液应在黑暗或光线较暗的开展 。 幻灯片不应该被允许干，直到最后一步。 <ol><li>设置之一：1）宽充满清洗液＃1 @ 53 ° C，和4个滑动洗碗充满的每250毫升以下的Coplin JAR灰解决方案＃1 @ 53 ° C，2）洗涤液＃2 @室温，3＆4）​​两道菜含有清洗液＃3 @室温。 </li><li>小心取出杂交商会的幻灯片，并将其放置在到Coplin JAR他们两个。离开30秒，然后抬起幻灯片慢慢和升降机滑仍将在罐子。放置幻灯片在幻灯片架，并立即浸入到清洗液＃1（1X SSC，0.2％SDS，预热到43 ° C）。重复，直到所有LifterSlips被删除，所有要洗的幻灯片，在洗涤液＃1碟。投身机架10倍上下大力。 </li><li>地点菜在53 °空中摇晃10分钟的转速在50-100摇床中的C。 </li><li>传输机架在室温下清洗解决方案＃2（0.1X SSC，0.2％SDS），暴跌10倍，并培育，缓慢摇晃10分钟。 </li><li>浸机架到先洗解决方案＃3（0.1X SSC）的15倍，以消除任何残留的SDS进行清洗液＃2。第二次洗涤液放置在机架＃3盘，大跌10倍，然后缓慢摇晃10分钟。 </li><li>配备摆动桶转子放置在50毫升猎鹰上限管和离心旋转@ 1500转幻灯片。删除幻灯片光隔音容器，我们使用另一个猎鹰管，用铝箔覆盖，并清除管内的空气与氮气，紧紧第（这样可以减少在夏季氧化的Cy染料，特别是CY - 5时，臭氧的水平最高的）。幻灯片可以存储在扫描中的氮清除管前几个小时，然而，获得最佳的信号效果，清洗后立即进行扫描时。 </li></ol>第9部分：数据收集和统计分析<ol><li>扫描图像数据的收集上珀金埃尔默ScanArray。我们使用10微米扫描设置和平衡Cy3标记和Cy5信号使用为制造商的指示线的调整方法。 </li><li> TIF文件是网格和原始信号数据的收集和筛选ImaGene版本7.5（生物发现，公司，El Segundo的，CA） </li><li>数据标准化和评估，以确定有效的统计差异表达的物种与BRB阵列工具版本。 3.7。 </li><li>模式分析，以方便协调基因表达的决心，是与基因表达模式分析软件（GEPAS）版。 3.1。 </li></ol><img src="/files/ftp_upload/1182/Figure1_WWLorenz-1.png" alt="图1" /> 图1 PtGen2处理数组使用此protoco 湖的例子 。 <img src="/files/ftp_upload/1182/Figure2_WWLorenz.png" alt="图2" /> 图2：使用标准的杂交和洗涤协议处理PtGen2数组的例子。

<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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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Pre-hybridization Buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5X SSC, 0.1% SDS, 1% BSA</td>
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<tr>
<td>Hybridization Buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>30% formamide, 5X SSC, 5X Denhardt&#x2019;s, 1% PolyA RNA (Invitrogen, POLYA.GF), 0.1% SDS</td>
</tr>
<tr>
<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>
</tr>
<tr>
<td>Wash Solution #2</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.1X SSC, 0.2% SDS</td>
</tr>
<tr>
<td>Wash Solution #3</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.1X SSC</td>
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<tr>
<td>Isopropyl Alcohol</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>34959-2.5L</td>
<td>&nbsp;</td>
<tr>
<td>50mL Conical Tubes</td>
<td>Other</td>
<td>Falcon&#x2122;</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>15mL Conical Tubes</td>
<td>Other</td>
<td>Falcon&#x2122;</td>
<td>352196</td>
<td>Use this or a similar cap placed at the bottom of each 50mL conical tube during centrifugation</td>
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<tr>
<td>Coplin Jar</td>
<td>&nbsp;</td>
<td>Wheaton&#x2122;</td>
<td>900520</td>
<td>&nbsp;</td>
<tr>
<td>Staining Dish & Rack </td>
<td>Other</td>
<td>Wheaton&#x2122;</td>
<td>900200</td>
<td>&nbsp;</td>
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<td>Albumin from bovine serum (BSA)</td>
<td>Other</td>
<td>Sigma&#x2122;</td>
<td>A-9418</td>
<td>&nbsp;</td>
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<td>PolyA RNA</td>
<td>&nbsp;</td>
<td>Invitrogen&#x2122;</td>
<td>POLYA.GF</td>
<td>&nbsp;</td>
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<td>SuperScriptTM Indirect cDNA Labeling </td>
<td>Kit</td>
<td>Invitrogen&#x2122;</td>
<td>L1014-02</td>
<td>&nbsp;</td>
<tr>
<td>Turbo DNase</td>
<td>Kit</td>
<td>Ambion</td>
<td>AM1907</td>
<td>&nbsp;</td>
<tr>
<td>System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 Dye</td>
<td>Reagent</td>
<td>GE&#x2122;</td>
<td>PA25001</td>
<td>&nbsp;</td>
<tr>
<td>Cy-3 Dye</td>
<td>Reagent</td>
<td>GE&#x2122;</td>
<td>PA23001</td>
<td>&nbsp;</td>
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<td>LifterSlips&trade;</td>
<td>Other</td>
<td>Erie Scientific Company&#x2122; </td>
<td>25X601</td>
<td>&nbsp;</td>
<tr>
<td>HybChambers</td>
<td>Equipment</td>
<td>GeneMachines</td>
<td>JHYB200003</td>
<td>&nbsp;</td>		</tbody>
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		</div>

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 ...

processing-the-loblolly-pine-ptgen2-cdna-microarray

Research

JoVE Journal

Biology

7.3K 次观看.  University of Georgia (UGA). 的cDNA微阵列PtGen2的基因表达研究开发火炬松， P。火炬松，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。

视频: Processing the Loblolly Pine PtGen2 cDNA Microarray

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae子囊孢子生产和收集微阵列实验。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

科研

生物学

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

的RNA分离方法的改进火炬松（ P。火炬松 L）和其他针叶树种

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Performing Custom MicroRNA Microarray Experiments

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

执行自定义的微RNA基因芯片实验

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.