这项工作是支持由教育署补助詹姆斯S麦克唐纳基金会21 世纪的奖励计划，和一个美国国立卫生研究院主任的新的创新奖。 GAB支持部分由加州再生医学研究所的博士后研究。

1。 2100生物分析仪的RNA样品的质量控制分析在开始之前， <ol><li>变性梯子，你的样品在70℃10分钟。冰寒立即在冰上。 </li><li>清洁与RNaseZAP 1分钟的工具电极，无RNase水为10秒。允许风干的电极。 </li><li>凝胶基质配制成试剂盒提供了一个自旋过滤器pipeting 550μL凝胶基质。在1500离心力离心机在室温为10分钟。分装过滤长达1个月到65μL分装和储存在4 ° C的凝胶。 </li><li>要准备的芯片吸站， <ol class="lalpha"><li>通过剪辑和到鲁尔锁适配器插入一个1 ml注射器。 </li><li>调整底座基地螺丝松动，升降板，拧紧螺丝位置的彗星。 </li><li>调整注射器的剪辑顶端的位置。 </li></ol></li><li>平衡至室温的染料集中。涡为10秒。染料加入1μL到65μL过滤凝胶等分。涡和13000离心力离心10分钟在室温。在一天之内使用。 当您准备好运行你的样品， </li><li>芯片上的吸站的位置。在这个协议中，我们使用的是RNA的6000纳米的芯片。要加载的凝胶， <ol class="lalpha"><li>吸取9μL凝胶染料混合在黑色背景与白色的“G”标记。确保你的小费是定位在井的最底部。 </li><li> 1毫升的注射器柱塞的位置。关闭吸站。确保点击它锁。 </li><li>按柱塞下行缓慢但稳步地，直到它被剪辑举行。 </li><li>等待30秒。释放剪辑。 </li><li>让柱塞本身。它停止移动后，等待几秒钟，柱塞回拉至1毫升的位置。打开吸站。 </li></ol></li><li>吸取9μL凝胶染料混合在每一个标有“G”的2口井。 </li><li>吸取5μL中的12个样品孔中的每一个阶梯以及标记。 </li><li>吸取1μL准备在阶梯阶梯。 </li><li>吸取1μL，每个样品的进样口井。 </li><li>吸取1μL在每个未使用的示例的标志。 </li><li>涡旋芯片为2400 RPM 1分钟。 </li><li>芯片运行， <ol class="lalpha"><li>启动2100专家软件。 </li><li>芯片的位置，并盖上盖子。如果仪器上线，图标将显示盖子是否打开或关闭，什么类型的芯片插入。确保选择了正确的端口。 </li><li>运行加载样本，以防止水分蒸发的5分钟内检测。 </li></ol></li></ol> 2。扩增和标签要准备微阵列分析，RNA样品扩增和标记，通常基于T7 RNA聚合酶3的反应。双链cDNA的是由反转录产生的。基因是用于在体外转录反应生成什么是已知的cRNA的。这种反应是在标记核苷酸的存在，生产微克阵列杂交标记的RNA数量。放大/标签方法的选择取决于对后续要使用的芯片平台。在本节中，我们描述了荧光标记的RNA的产生，使用安捷伦的快速放标记试剂盒。 <ol><li>吸取50毫微克至5微克的RNA在1.5毫升管。量应不超过8.3μL。如果有必要，集中力量真空离心或用酒精沉淀的RNA样品。 </li><li>加入1.2μL的T7引物。 </li><li>带来的最终体积11.5μl无RNase水的。 </li><li>孵育在65 ° C下10分钟变性的RNA。我们推荐使用一个热循环与热盖，以防止在管的顶部结露。 </li><li>在10分钟的孵化，温暖的5X第一链缓冲到80 ° C为5分钟。涡和自旋向下。在室温下保存，直到准备使用。 </li><li>经过10分钟的孵化，迅速降温变性的RNA样品在冰上。 </li><li>准备主结构的cDNA。每个反应中，添加以下内容： <ol class="lalpha"><li> 4μL5X第一链缓冲</li><li> μL0.1 M的数码地面电视</li><li> 1μL10 mM的dNTPs </li><li> 1μLMMLV逆转录酶</li><li> 0.5μLRNase的输出。 </li></ol></li><li>混合组件的反相管的4倍。不要旋涡。离心收集管底部的内容。 </li><li>掌握每个样品的混合添加8.5μL。混合向上和向下通过pipeting。 </li><li>孵育2小时40℃，其次是10分钟在65 ° C。我们建议使用一个热循环加热的盖子。 </li><li>管转移到冰浴5分钟。 </li><li>准备转录预混。每个反应中，添加以下内容： <ol class="lalpha"><li> 15.3μL无核酸酶水</li><li> 20μL4X转录缓冲液</li><li> 6μL0.1中号数码地面电视</li><li> 8μLNTPS </li><li> 6.4μL50％聚乙二醇（PEG应预热至40℃和振荡，以确保再悬浮） </li><li> 0.5μLRNase的输出</li><li> 0.6μL无机焦磷酸</li><li> T7 RNA聚合酶0.8μL </li><li> 2.4μLCy3标记的CTP或Cy5 </li></ol></li><li>简要旋转的样品收集管底部的内容。 </li><li>转录预混每个样品添加60μL。 </li><li>在40 ° C孵育2小时。 </li><li>加入20μl无RNase水的。 </li><li>净化标记的cRNA消除非法人核苷酸。我们建议采用Qiagen公司的RNeasy迷你列。 </li><li>量化标记的cRNA。我们建议使用微阵列测量模式NanoDrop2000分光光度计。确保你选择样本类型的RNA - 40。 </li><li>记录样品的浓度，收益率（由量的浓度乘以计算）和具体的活动（1000乘以以上的cRNA浓度的染料浓度的口粮计算）。芯片杂交，实现了至少825 ng和一个特定的活动，至少8 pmol /微克的产量是必要的。 </li></ol> 3。芯片杂交<ol><li>打开杂交站，并设置在65 °彗星至少2小时前开始杂交。 </li><li>要准备杂交样本，混合在1.5毫升管以下内容： <ol class="lalpha"><li> 825吴Cy3标记的cRNA（这通常是你的“待遇”样本） </li><li> 825吴Cy5的cRNA的（这通常是你的“参考”样本） </li><li> 11μL10X阻断剂。 </li><li>加入无RNase水到终体积为52.8μL </li><li>添加2.2μL碎片缓冲。 </li></ol></li><li>在低速漩涡混合。 </li><li>在60℃孵育整整30分钟。这是cRNA的碎片的步骤，生成阵列杂交标记的片段。这是非常重要的孵化完全描述。我们建议使用一个与热盖的热循环。这是非常重要的孵化完全描述生成正确的平均长度的探针。碎片过多或不足会导致漏报或误报。 </li><li> 2X杂交缓冲液中添加55μL停止碎片。混合吹打，特别注意不要引入气泡。 </li><li>最高速度为1分钟，离心收集在试管底部的内容。如果观察气泡，离心机更长的时间将其删除。 </li><li>将冰管，轻保护。尽快负载阵列上的样品。 </li><li>要加载阵列，首先准备杂交室。这个协议描述安捷伦4x44K阵列使用罗氏NimbleGen的杂交系统和A4搅拌机商会。 <ol class="lalpha"><li>首先在芯片组装/拆卸工具，条码侧幻灯片。 </li><li>打开一张A4混频器和暴露的粘合剂。控股阵列和组装/拆卸工具，以防止任何运动，在幻灯片上放置A4的混频器，在远端开始。确保它是正确对齐的工具。坚持沿阵列幻灯片混频器。 </li><li>从混频器的结束，拉出幻灯片/混频器组件。 </li><li>使用工具的叫声，按沿粘合垫片，以确保它紧紧地粘在theslide。小型空气被困之间的粘合剂和幻灯片的气泡可以看到黑暗背景。花额外的时间来清除这些气泡叫声工具。现在已经准备好要加载您的阵列。 </li></ol></li><li>使用正位移移液器负载100μL杂交样品。按提示紧紧端口孔，然后免除缓慢而平稳地使空气不被困在数组区域。永远不要尝试吸的样品备份。一旦整个样本已经被取消，不释放柱塞端口上的洞的一角。当样本覆盖了整个数组和液体开始走出来的宣泄口，迅速取出吸管。只释放柱塞一次提示是远离幻灯片。 </li><li>轻轻民建联在两个端口多余的液体。确保你不画分庭本身的样本。 </li><li>与贴纸端口孔盖，用小镊子。 </li><li>杂交站上的幻灯片，检查膀胱孔O型环，正确定位。这将确保在杂交适当的混合。 </li><li>关闭滑盖和车站盖。设置混合模式B站</li><li>杂交阵列在65 ° C为17小时。 </li><li>准备洗净TRITON X - 102加入到终浓度为0.005％的缓冲区2。保持在37℃过夜。 </li></ol> 4。芯片洗<ol><li> TRITON X - 102洗涤缓冲液1到终浓度为0.005％。保持房间temperaturE. </li><li>杂交完成后，填写一个玻璃盘，用洗涤缓冲液1的“A”。这道菜应该大到足以容纳组装/拆卸工具，以及允许一些机动。所有清洗工作应在玻璃器皿。避免塑料菜，因为他们往往​​在阵列中的高背景产生的浸出化合物。 </li><li>放幻灯片染色菜的“B”的机架和一个搅拌棒。填充洗涤液1，确认它涵盖了机架。放置在室温下搅拌板。 </li><li>放在一个空盘染色的“C”和不小的轰动板一个搅拌棒。 </li><li>删除数组，并把它在组装/拆卸工具。淹没在盘子里的“A”整个大会。 </li><li>按住两侧的装配/拆卸工具和幻灯片，用一只手，仔细剥离的A4混频器。请务必不要划伤阵列面积。 </li><li>快速放置在机架染色菜的“B”的幻灯片。从现在起，应尽量减少暴露在空气中，由于Cy5的染料敏感臭氧。不要洗一次超过8阵列。 </li><li>用中等速度为1分钟的搅拌。 </li><li>填充染色的“C”盘预热的缓冲液洗2。传输幻灯片和洗1分钟。 </li><li>非常缓慢去除染色盘的“C”，以最大限度地减少在幻灯片上留下的水滴滑动机架。 </li><li>干燥2分钟旋转的幻灯片。如果不兼容的离心机，离心机在50毫升的锥形管的幻灯片或吹氩气。 </li><li>在50毫升的锥形管放置幻灯片和填充氩气。扫描立即以避免信号损失。我们建议使用GenePix 4000B扫描仪从分子器件。 </li></ol> 5。代表性的成果： 良好的质量总RNA样品应只有两个主要峰时，在生物分析仪的质量控制，相应的2个主要的核糖体RNA物种。一些RNA降解，将显示作为涂抹核糖体RNA前第一高峰。严重退化，低质量的RNA将呈现一个宽峰或一系列峰低的保留时间，而核糖体RNA峰强度极低或无法识别所有。 2100生物分析仪输出的例子，见图1。 专家2100软件将计算RNA的完整性号码，或RIN，作为RNA质量的定量测量。高品质样本的RIN值高于9，显然是最好的芯片应用。但是，我们使用的RIN值低5.2，在合理的质量产生的微阵列数据样本。 质量好的阵列产生高信号，在相对低的光电倍增管值。在我们的实验中，大多数的成绩单，预计在类似的实验和参考样本水平目前，大规模的广泛的变化，在基因的表达，可能会没有任何生物学意义。因此，数组最应该看黄色而非绿色或红色。良好的信号质量也应在动态范围的信号直方图完全重叠。举例来说，见图2。 <img src="/files/ftp_upload/2546/2546fig1.jpg" alt="图1" /> 图1。四个RNA样品从人脑肿瘤组织中分离的例子。 RNA的提取，使用3.1.1中提出的协议。 2100生物分析仪上使用的RNA 6000第3.1.3条所述的芯片样品的质量进行了评估。样品代表4个不同的RNA的素质，很好的RNA的质量（RIN&gt; 9）; B，部分降解的RNA（RIN的5-6，注意28S rRNA的峰值显着降低），C，高度降解的RNA（RIN &lt; 3），D，几乎完全降解RNA（RIN = 2）。我们已经成功地使用相似或比B（RIN&gt; 5.2），更好地为下游芯片的应用与素质的样品。固体和开放的箭头指示的位置，分别为18S和28S rRNA的物种。 <img src="/files/ftp_upload/2546/2546fig2.jpg" alt="图2" /> 图2。阵列图像和散点图的例子。一，整个幻灯片预览，显示在4X44K安捷伦格式的四个阵列，B，高分辨率扫描阵列的领域之一，请注意，没有任何信号（红色或绿色）是主要的，分散的图像绘制在B，注意的信号完全重叠和不超过1 × 10 -6的特点是饱和强度。

<ol>
	<li>Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+Decade+Later:+What+has+Gene+Expression+Profiling+Told+us+About+Neuronal+Cell+Types,+Brain+Function+and+Disease.">One Decade Later: What has Gene Expression Profiling Told us About Neuronal Cell Types, Brain Function and Disease.</a> Curr Genomics. 10, 318-318 (2009).</li><li>Barisone, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Expression profiling in Neuroscience, edited by Ioannis Karamanos. , (2010).</li><li>Gelder, R. N. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc Natl Acad Sci U S A. 87, 1663-1663 (1990).</li></ol>

表达与DNA微阵列分析提供了一个简单的方法来确定两个生物样品之间的差异表达基因。成功的表达谱实验需要高品质的RNA样品，强大的标签和杂交。根据我们的经验，安捷伦的商业阵列和标记试剂盒，在合理的成本提供高质量的数据。安捷伦还提供自己的杂交和扫描机器，我们赞成罗氏NimbleGen的GenePix 4000B芯片扫描仪从分子器件的杂交系统。需要注意的是，罗氏NimbleGen的杂交单位前身为MAUI杂交系统。最近由罗氏公司收购后，已经停产的A4混合器。截至写这篇文章的时刻，他们仍然可以通过Kreatech诊断（如其他卖家<a href="http://www.kreatech.com/">www.kreatech.com</a> ;这个网站还提供了一个方便阵列的兼容性的工具）被发现，但长期的库存量是不确定的。其他杂交系统（例如，从安捷伦）;但是，如果兼容的混频器，可为正在使用的阵列，我们建议罗氏NimbleGen的系统，其质量和可重复性。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2100 Bioanalyzer</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G2943CA</td>
<td>Includes chip priming station, vortexer, software and laptop computer</td>
</tr>
<tr>
<td>2100 Bioanalyzer electrophoresis set</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G2947CA</td>
<td>&nbsp;</td>
<tr>
<td>RNA 6000 Nano kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5067-1511</td>
<td>includes size ladder, marker, gel matrix, dye, electrode cleaners, spin filters and nanochips</td>
</tr>
<tr>
<td>RNase Zap</td>
<td>&nbsp;</td>
<td>Applied Biosystems &#x2013; Ambion</td>
<td>AM9780M</td>
<td>&nbsp;</td>
<tr>
<td>Quick Amp labeling kit, two-color</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5190-0444</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy mini kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74104</td>
<td>&nbsp;</td>
<tr>
<td>Gene Expression Hybridization Kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5188-542</td>
<td>Includes Blocking agent, Fragmentation buffer and Hybridization buffer</td>
</tr>
<tr>
<td>Gene Expression Wash Buffer Kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5188-5327</td>
<td>Includes Wash Buffers 1 and 2 and Triton X-102</td>
</tr>
<tr>
<td>Hybridization Station</td>
<td>&nbsp;</td>
<td>Roche Nimblegen</td>
<td>05223652001</td>
<td>4-slide model</td>
</tr>
<tr>
<td>Hybridization System Accesory Kit</td>
<td>&nbsp;</td>
<td>Roche Nimblegen</td>
<td>05327695001</td>
<td>Contains verification assembly for testing mixing, replacement O-rings, disassembly tool, forceps and brayer</td>
</tr>
<tr>
<td>A4 mixer hybridization chambers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Whole Human Genome (4x44) Oligo Microarray</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G4112F</td>
<td>&nbsp;</td>
<tr>
<td>Positive displacement pipette</td>
<td>&nbsp;</td>
<td>Gilson</td>
<td>F148504</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pistons for positive displacement pipette</td>
<td>&nbsp;</td>
<td>Gilson</td>
<td>F148415</td>
<td>&nbsp;</td>
<tr>
<td>Microarray dryer</td>
<td>&nbsp;</td>
<td>Nimblegen</td>
<td>05223636001</td>
<td>&nbsp;</td>
<tr>
<td>Microarray fluorescent scanner, Axon GenePix 4000B</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>GENEPIX4000B</td>
<td>&nbsp;</td>
<tr>
<td>GenePix Pro 6.0 software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna microarrays: sample quality control, array hybridization and scanning

芯片表达谱的神经系统提供强大的方法，以确定在不同发展阶段，不同的生理或病理状态，对治疗的反应，的基因活动，并在一般情况下，任何正在实验测试条件。神经组织中的表达分析需要的高质量的RNA，分离出的RNA和DNA芯片杂交扩增隔离。在这篇文章中，我们描述了从脑肿瘤组织 2重复的微阵列实验的协议。我们将开始执行一个孤立的RNA样品的质量控制分析与安捷伦2100生物分析仪“实验室的单芯片”技术。高品质的RNA样品的任何芯片实验成功的关键，和2100生物分析仪提供了一种快速，定量测量样品的质量。 RNA样品，然后放大，并通过执行反转录获得cDNA标记，在体外转录标记的核苷酸的存在产生标记的cRNA。我们将通过使用一个双色标记试剂盒，标签我们的实验样品，用Cy3与Cy5的参考样本。两个样本，然后将结合安捷伦的4x44 K阵列杂交。双色阵列提供了两个RNA样品之间的直接比较的优势，从而提高了测量的精度，特别是为表达水平的小变化，因为这两个RNA样品杂交竞争力的一个单一的芯片。阵列将在两个对应的波长进行扫描，并使用Cy3标记，以Cy5的信号为每个功能的比例将作为相应的mRNA的相对丰度的直接测量。这种分析确定，在被测试的实验条件的差异表达基因。

Watch this Scientific Journal Video about DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning at JoVE.com

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

我们展示了利用DNA芯片为表达对神经系统的分析。我们描述了RNA的质量控制，样品标签，和阵列杂交和扫描。

DNA微阵列：样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

dna-microarrays-sample-quality-control-array-hybridization

Research

JoVE Journal

Biology

37.8K 次观看.  University of California, Davis. 我们展示了利用DNA芯片为表达对神经系统的分析。我们描述了RNA的质量控制，样品标签，和阵列杂交和扫描。

视频: DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全基因组阵列比较基因组杂交技术示范

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

科研

生物学

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

制造和使用微环境芯片（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

该做什么和不该做什么低温电子显微镜的：入门样品制备和高质量的数据采集为大分子三维重建

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...