这项工作的一部分，由美国国立卫生研究院（NIH）资助R 44 AI072784支持。我们感谢基尔斯滕圣乔治博士，萨拉B. Griesemer，达里尔·拉姆森和沃兹沃斯中心，纽约州卫生部门量化的流感病毒粒子进入临床验证流感实时病毒病，实验室的艾米院长院士PCR检测。

三的自动化TruTip提取协议描述，这里演示:1）中等通量RNA提取的Eppendorf epMotion 5070 NPA 2）高通量基因组DNA提取从全血汉密尔顿STAR量低，和3）选择性分离母体血浆量汉密尔顿的Starplus上从大的支离破碎的胎儿DNA富集。自动的脚本能从Akonni和只有高级程序描述符在这里提供。的提取和洗脱试剂都是自动从批量试剂槽分配到96孔板中的自动脚本（次）前处理的临床样品。 1。自动RNA提取鼻咽分泌物先前所描述的的手动TruTip方法15现在适应的Eppendorf epMotion 5070液体处理机器人上运行，使用1.0毫升的Eppendorf管中的的大孔隙TruTip矩阵嵌入式TTE提示，2毫升的深孔板（美国科学），Akonni的提取TruTip试剂，作为样品类型和NPA。 epMotion 5070液体处理机器人最多可容纳8个技巧的同时，使基线自动化协议描述为8位并行提取。然而，可以处理多达24个样品，在一个单一的程序在一个深水井96孔样品板。一个单独的epMotion程序（和）以16或24个样品进行处理。下面列出的是该协议8自动化脚本示例。 格局<ol><li>鼻咽样本带来RT开始前提取。 </li><li>分配100μL鼻咽分泌物加150无核酸酶的水倒入样品板（ 图2A）第1栏。 </li><li>将样品板到位置B1上的的epMotion工作台（ 图2B）。 </li><li>将枪头，的TruTips和30毫升试剂槽到各自的epMotion Worktab的中的文件的位置（ 图2B）。 </li><li>打开的Eppendorf epBlue软件，选择"运行文件由Akonni提供8个样品，并点击运行按钮"运行"选项卡上加载的方法。 </li><li>根据液位传感器设置，选择水平和技巧，然后单击"运行"按钮。 </li><li>到软件输入样品的体积，然后单击"运行"。 </li><li>该的epMotion脚本将提示用户添加萃取和洗脱试剂试剂水库位于工作台位置B2。添加每种试剂的推荐量到各自的低谷。对于8个样品中，最小的试剂量是: </li></ol><table border="1"><tbody><tr><td> 试剂 </td><td> 体积（ml） </td><td> 海槽位置 </td></tr><tr><td> 95％乙醇</td><td> 3.5 </td><td> 2 </td></tr><tr><td>洗涤缓冲液D </td><td> 9 </td><td> 3 </td></tr><tr><td>洗涤液ê </td><td> 9 </tD&gt; <td> 4 </td></tr><tr><td>洗脱缓冲A2的</td><td> 1.3 </td><td> 5 </td></tr><tr><td>裂解并绑定缓冲液D </td><td> 11.0 </td><td> 6 </td></tr></tbody></table><ol start="9"><li>输入试剂卷epMotion软件时提示从上面的步骤8成表。输入值应反映到每个相应的试剂槽由用户分配的缓冲区的实际体积，必须大于或等于上面提到的最小体积。卷项不正确，可能会导致不正确的体积在96 - 孔板（次）由的epMotion硬件的每个管或交付。 </li></ol>自动化程序<ol start="10"><li>选择RUN启动自动化的方法。自动化脚本将移动没有用户干预的情况下通过以下步骤进行: 裂解样品和试剂分配: <ol><li>免除375μL裂解缓冲液D 1列混合10的周期（apirate +免除= 1周期）。这一步开始裂解孵化过程中，而其余试剂等分。 </li><li>免除1.6毫升的洗涤液D进入第2列。 </li><li>免除1.6毫升的洗涤液E组成3栏。 </li><li>免除50μL洗脱缓冲液A进入第4列。 </li><li>暂停6分钟完成10分钟取样培养裂解液D. </li><li>在第1列，每孔加入375μL乙醇，通过与乙醇混合每个样品10个吹打周期。 </li></ol> 提取: <ol start="7"><li>将装入8 TruTips从位置A2工作台，并开始提取过程如图1所列。 </li><li>吸入和排出样品/裂解缓冲/乙醇混合物的样品板的第1列的七个周期绑定的核酸的TruTip巨石。虽然样品通过TruTip流可能有所（由于临床样品的粘度差异），这些样品的核酸产量没有吨流量变化的影响。在讨论提高样品流的选项。 </li><li>将移动TruTips样板柱2，循环洗涤缓冲液D 5x的过巨石，以去除残留的裂解液样品基质。 </li><li>将移动TruTips样板柱3，循环洗涤缓冲液ê5x的过巨石去除蛋白质结合的核酸和其他污染物。 </li><li>将移动TruTips空试剂瓶位置1（在工作台位置B2），和周期80X（在流速快）干的巨石。重要的是要彻底干燥的TruTip，洗脱的核酸制剂中的残留溶剂将产生负面影响酶，如逆转录酶和Taq DNA聚合酶。 </li><li>移动TruTips样板柱洗脱缓冲A. 4和周期的5倍，提取和纯化核酸是现在在洗脱缓冲液中样品板式塔4口井。 </li><li>的epMotion垃圾桶中弹出TruTips"。 </li><li&gt;当程序完成后，以手动方式从仪器中取出的样品板和纯化的核酸转移到新的管中，用于长期贮存或进一步使用。高级的epMotion用户可以添加指令运行文件转移到单独的存储管或PCR板块的洗脱样品，如果需要的话。 </li></ol></li></ol>总共16个样本的程序将重复步骤10.1至10.13用样品板列5-8。对于24个样品的程序，步骤10.1重复通过10.13 2X使用样板5-8和9-12，分别列。 2。 96从全血中提取基因组DNA 汉密尔顿STAR液体处理机器人可用于同时显示96个样品的自动提取，从全血中。哈密​​尔顿星不同于的epMotion系统的一个可选的加热器/摇床单元可在甲板上，这是重要的临床目标酶消化校准矩阵，如全血。一个96通道的移液管头，因为该系统可以配备有一个专用的96孔板的每个的TruTip的步骤和试剂。 格局<ol><li>打开仪器和计算机上的STAR。 </li><li>打开汉密尔顿运行控制软件。 </li><li>打开运行文件96个样品Akonni。 </li><li>将实验器具的STAR甲板上， 如图3所示。 </li><li>免除到相应的槽（卷表示至少需要处理96个样品）试剂: </li></ol><table border="1"><tbody><tr><td> 试剂 </td><td> 体积（ml） </td><td> 海槽位置 </td></tr><tr><td>裂解和结合缓冲液F </td><td> 75 </td><td> 5 </td></tr><tr><td> 95％乙醇</td><td> 100 </td><td> 6 </td></tr><tr><td>洗涤液Ĵ </td><td> 175 </td><td> 7 </td></tr><tr><td>洗缓冲区K表</td><td> 175 </td><td> 8 </td></tr><tr><td>洗脱缓冲A2的</td><td> 12 </td><td> 9 </td></tr><tr><td>蛋白酶K（20毫克毫升-1） </td><td> 8 </td><td> 15 </td></tr></tbody></table> 6。允许样品平衡至RT。 7。将样品管样品载波架（甲板图3中的位置4）。样品1的背面最左边的载体，并依次向下移动每个载波样品96的结局在右前方的位置。 自动化程序<ol start="8"><li>选择运行文件左上角的窗口和输入的数量正在处理样品中的播放按钮。自动化的脚本，然后移动没有用户干预的情况下通过以下步骤进行: 样品溶解和试剂分配 <ol><li>从每个样品管中加入200μl转移到培养板，在第14位上的他亚特/:摇床模块（ 图3）。 </li><li>分配80μL蛋白酶K孵化板到每个样品。 </li><li>在进入孵化板每孔600μL裂解缓冲液F免除。 </li><li>混合溶液进行10个循环，然后在70℃和500rpm下孵育20分钟。的70℃的设定温度在〜60℃下的样品的温度在深孔板蛋白酶K的活性的范围内，这是内。虽然样品孵化，液体处理系统继续工作由分配到相应的板块和水井试剂: <ul><li>加入100μl洗脱缓冲液A到13位的深孔板的各孔中。 </li><li>到10位的深孔板的各孔中的800微升乙醇。 </li><li> 1.6毫升的洗涤缓冲液Ķ到位置12深孔板的各孔中。 </li><li> 1.6毫升的洗涤缓冲液Ĵ到11位的深孔板的各孔中。 </li></ul></li><li孵育20分钟后，从培养板的样品混合物转移到10位的深孔板，并通过移液12次混合。 </li><li>弹出试剂提示垃圾桶。 萃取 此部分的基因组DNA的血过程是非常相似的的epMotion流感协议，除了洗涤组合物，试剂和循环数。汉密尔顿TruTips提示是黑色的，所以液体的流动通过TruTip是不可见的。 </li></ol><ol start="7"><li>将96 TruTips从甲板位置3。 </li><li>吸入和排出的样品/裂解缓冲/乙醇混合物于10周期结合核酸到TruTip巨石位置10。 </li><li>将TruTips移动到位置11和循环洗涤缓冲液Ĵ5x的过巨石，以去除残留的裂解液样品基质。 </li><li>移动TruTips的位置12和循环洗涤缓冲液K表5倍，去除蛋白质和其他污染物结合的核酸。 </li&gt; <li>循环TruTip 40X高速风干。 </li><li>将移动TruTips位置13和周期在洗脱缓冲A2 5倍。的提取和纯化的核酸是现在在洗脱缓冲液中的深孔板。 </li><li>垃圾桶弹出TruTips。 </li></ol></li></ol>当程序完成后，取出洗脱从仪表板和提取的样品转移到相应的管存储或下游应用。 3。大体积的血浆样本的DNA提取汉密尔顿的Starplus仪器是用来证明自由提取循环从5毫升母体血浆胎儿DNA的自动化协议。 STARPLUS系统可以支持两种自动移液器通道臂，一个8×5毫升渠道和8×1毫升渠道之一。这些武器可以并行工作，在每个批次的8个样品进行交错处理。 5毫升TruTip用于初始升阿尔赫体积萃取，和1毫升TruTip的用于规模分离和进一步浓缩的提取的核酸。 设置<ol><li>打开STARPLUS仪器和计算机。 </li><li>打开汉密尔顿运行控制软件。 </li><li>打开运行文件由Akonni提供8大体积的血浆样本。 </li><li>如在图4中所示将实验器具到的Starplus的甲板上。 </li><li>免除到相应的水库试剂: </li></ol><table border="1"><tbody><tr><td> 试剂 </td><td> 体积（ml） </td><td> 海槽位置 </td></tr><tr><td> CN-W1 </td><td> 17 </td><td> 5A </td></tr><tr><td> CN-W2 </td><td> 17 </td><td> 5B </td></tr><tr><td> CN-W4 </td><td> 21 </td><td> 5C </td></tr><tr><td>蛋白酶K（20毫克毫升-1） </td><td> 5 </td><td> 6A </td></tr><tr><td> EBB </td><td> 17 </tD&gt; <td> 6B </td></tr><tr><td> EBA2 </td><td> 5 </td><td> 6C </td></tr><tr><td> CN-W3 </td><td> 5 </td><td> 6D </td></tr><tr><td> CN-B2 </td><td> 5 </td><td> 6E </td></tr><tr><td> CN-B3 </td><td> 5 </td><td> 6F </td></tr><tr><td> CN-L1 </td><td> 52 </td><td> 7 </td></tr><tr><td> CN-B1 </td><td> 175 </td><td> 8 </td></tr></tbody></table><ol start="6"><li>允许样品平衡至室温。 </li><li>将样品管样品载波架（甲板位置3 图4）。将样品1在后方朝向前方的甲板上顺序地移动。 </li></ol>自动化程序由于大的输入的样品体积，必须进行裂解和均质化步骤，在水浴中汉密尔顿的Starplus仪器。步骤无需用户干预的自动化协议内有星号（*）表示在搁克的句子，和大胆的类型 。 裂解样品和试剂分配:样品用蛋白酶K和裂解缓冲液中温育的样品均匀化，并释放DNA。 <ol start="8"><li>选择左上角的"运行文件"窗口中的播放按钮。正在处理的输入的样本数，在甲板上的移液吸头的位置，在甲板上的位置TruTips。 </li><li>的自动脚本。添加615微升的蛋白酶K，5毫升血浆，和6.2毫升的裂解缓冲液CN-L1每次50毫升锥形管，然后将PAUSE（暂停）。 </li><li> *从样品甲板，旋涡中取出50毫升锥形管高速30秒，脱机，孵育30分钟，在60℃水浴或者加热块。锥形管后从汉密尔顿甲板，删除恢复的自动化脚本将继续分配到各自的板和井（ 图4B及4C）试剂: <ul&gt; <li> 2毫升CN-W1以及所有其他位置的9列1。 </li><li> 2毫升CN-W2以及所有其他位置9列2。 </li><li> 2毫升CN-W4以及所有其他位置9 3栏。 </li><li> 250微升EBA2的每隔以及在第9位4和5栏。 </li><li> 495微升CN-B2以及所有其他的在位置10列1。 </li><li> 1毫升EBB以及所有其他位置10列2。 </li><li> 500微升CN-W3以及所有其他位置10第3栏。 </li><li> 500微升CN-W4位置以及所有其他的10列4。 </li><li> 50微升EBA2的以及所有其他位置10第5列。 </li></ul></li></ol>因为每个样品使用邻井，自动化程序无需试剂以及深孔板在甲板位置9到每一个其他的5毫升通道太宽。机械手臂使用1毫升通道也需要使用其他每个试剂板以及排斥和浓度STEPS的协议。 胶试剂后，程序将会暂停。 <ol start="11"><li> *，60℃温育30分钟后，将50毫升锥形管在冰上5分钟。 </li><li> *返回到原来的位置50 ml锥形管内的样品载体机架在甲板位置4，和恢复的自动化脚本。 </li><li>添加12毫升结合缓冲CN-B1每个样品管中混合10倍。 </li></ol> 大体积提取:使用5毫升TruTips提取总DNA裂解血浆样本。 <ol start="14"><li>拿起从位置2大体积的核酸提取5毫升TruTips。 </li><li>周期样品mixture15时间在50毫升锥形管，在管的底部，并开始移动3毫米每个移液周期后。此步骤的总核酸到TruTip整体式绑定。 </li><li>深孔板将TruTips移动位置1，第6纵队和cy第一百1X洗涤液CN-W1。 </li><li>移动TruTips 9列2和CN-W2洗周期1X定位。 </li><li>将TruTips移动定位9列3和周期2个洗C​​N-W4。 </li><li>移动的定位TruTips 9列4和周期的40倍，在高速干结合矩阵。 </li><li>将TruTips移动的位置9的第5栏和周期的10倍，以洗脱结合的核酸从5毫升TruTips的。这是大体积洗脱＃1。 </li><li>将移动TruTips塔6，并与第二洗脱缓冲液的等分试样中重复的步骤。这是大体积洗脱＃2。 </li><li>转移洗脱＃2到9位第5列，结合它与洗脱＃1，并丢弃TruTips的。 </li></ol>从所提 ​​取的样本中，被删除的排斥与浓度:高分子量DNA和其它DNA是孤立并浓缩。 <ol start="23"><li>新增合并洗脱液从第22步到10柱1定位调匀10倍。 </li><li>拿起1毫升TruTips的从位置13和周期20倍的高分子量DNA绑定到单块。 </li><li>移动的的定位TruTips 10列2和5倍周期冲洗小费和删除超高分子量DNA。该的保留TruTips 13位尖架放回。 </li><li>用试剂提示位置12，加575μL结合缓冲CN-B3位置10列1和混合10倍的样品。 </li><li>如拿起从步骤25 TruTips，返回10柱1，周期20X绑定其余的从样品DNA 1毫升TruTip，定位。 </li><li>移动TruTips，定位10列4周期1X洗CN-W3删除任何剩余的抑制剂。 </li><li>移动的的定位TruTips 10第5列和CN-W4在洗涤周期1X冲洗残留盐CN-W3。 </li><li>提高位置TruTips超过10第5列和循环空气通过提示35X干巨石。 </li><li>移动的位置TruTips 10列6和循环在EBA2 10倍洗脱纯化，大小选择性泰德和浓核酸。 </li><li>丢弃TruTips。 </li><li>转移的从塔6到第11位的1.5 ml微量离心管中洗脱样品。提取的样品是准备用于存储或下游加工。 </li></ol>

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员工的Akonni生物系统公司，在这篇文章中所使用的生产材料。 本文的免费接入和生产赞助由Akonni生物系统公司

简单的的TruTip概念和工作流程（ 图1）使得它很容易适应，一些临床样本矩阵，输入样本量，液体处理系统的自动化，高效和有效。然而，应当认识到，临床样品中，每一个是唯一的，并会随一个到下一个的粘度，颗粒物，粘液，表面的污染物，微生物，和/或人类遗传背景。在临床样品中的组合物和预期用途的一种自动化TruTip的样品制备协议的预期的变化，因此，它可能是必要的修改某些步骤一个TruTip程序，在为了实现期望的结果为一个特定的样品类型。然而，无论样品类型，通常具有最重要的影响核酸的纯度和/或恢复的TruTip参数包括: <ol><li> 混合样品，用裂解缓冲液（和酒精）的同质化。的 TruTips重新尺寸，样品孔隙latively大均质化和液化是非常重要的高效的细胞裂解，随后的结合步骤到TruTip整料。均匀的和液化的溶胞产物，样品具有较高的流速，从而降低了整体的样品处理时间通过TruTip移动。大体积等离子体协议展示潜在用户彻底同质化和液化困难的样本（在线或离线），尽管输入大样本量。 </li><li>较慢的流速的流速。核酸结合或洗脱过程中，通常会导致更高的核酸产量，虽然在总的处理时间为代价的。较低的流速也可减少DNA剪切的程度。 </li><li> 周期数。愿望的最佳数量和分配周期取决于样品类型，总样本量和流速。 图1中的步骤1通常是在哪些周期numbeRS（流速），可能需要一些经验优化，代表一个更具挑战性的溶胞产物优化由于NPA粘度的范围内，来自不同患者的鼻咽分泌物（ 图5），如样品。 </li><li> 干燥完成的干燥TruTip整料是必须防止有机溶剂残留量，从纯化的核酸样品，抑制下游工序或测试用的共洗脱。因为没有通过离心或真空过滤干燥，TruTip，重要的是最大限度地提高在干燥步骤期间的流量和循环次数。有时，洗涤溶液中的残留液滴的干燥周期完成后，上的TruTip末端。哈密​​尔顿机器人具有的能力，以执行一个"前端触摸"的侧面以及释放液滴，从而确保了一种无溶剂的洗脱。 epMotion系统不具备此功能，但预冲洗总站在EL TruTipution缓冲区可以被编程来达到同样的效果。 </li></ol>由于几何形状，材料的枪头，和连接方法机器人通道武器是唯一的每个仪器制造商中，不同TruTip构造所需为每个液体处理系统。与核酸结合的能力（和洗脱效率）的TruTip整体尺寸（直径，厚度和孔隙尺寸），预期的任何固相萃取技术。厚（&gt; 4毫米）的矩阵可以被嵌入到1毫升TruTip的，以增加核酸结合容量为大体积的样品和/或跨特定TruTip格式的结合能力相等，有一个折衷之间的TruTip厚度和流速初始的结合步骤（在粗裂解物的存在下）。因此，有时是有利的嵌入到较大 ​​体积移液吸头的较大直径的整料的一种自动化协议的初始步骤（ 如&lt;/ em&gt;的5毫升汉密尔顿/ Akonni的TruTips大批量提取）。然而，由于液体处理机器人由厂家决定的的具体TruTip配置，我们不期望核酸TruTip跨液体处理平台，来自不同制造商的，或跨不同TruTip大小的收益率是相同的。 临床样品（定义）将含有显着数量的人类基因组DNA，除非它们被收购常无菌部位（ 如脑脊髓液）。有时，在人基因组DNA是理想的（如图6所示），而在其他应用程序中的人类DNA指不想要的基因组背景（ 图5）。背景DNA的存在下通常是没有问题的，因为只要核酸的总量不超过样品中的单块的结合能力，和背景的DNA可以作为一种载体，如果所需的靶核酸酸是本痕量。的高容量的血浆中提取协议（ 图7）的目的是隔离（分段）中存在的10-20倍过量的母亲的DNA，这是类似的样品的制备传染病测试的目的，除了胎儿DNA序列高度一致，并，只能区分高度特异性分子检测和/或尺寸歧视。在这种情况下，总循环DNA是孤立使用5毫升TruTip，和随后的高分子和低分子量的胎儿DNA的分离通过随后的结合和洗脱1毫升TruTip的结合缓冲液条件下通过改变。大小选择性分离和富集靶核酸的基础上他们的结合和洗脱属性二氧化硅巨石是比膜或体积排阻离心柱实现的一个显着不同的作用模式。尺寸分离和富集微生物DNA的人类基因组DNA可能是一个在未来的应用ccomplished，通过定制TruTip绑定和洗脱缓冲。 自动化的协议表明，这里强调的的巨石TruTip本身的效用，以及如何处理不同的临床样本，它可以适应大量特定的液体处理机器人。精简的方法通常会导致更快的提取协议相比其他自动化系统。然而，的简单的TruTip技术，也为那些有兴趣购买一个新的，自动化核酸纯化系统提供了一定的成本优势，因为需要用于自动化TruTip程序的主要硬件是移液器通道臂本身，而不是磁力棒，真空系统或板上离心机。利用预充试剂板也可以减少空间和所需的消耗品，每运行吞吐量翻番。甲板最小化空间与TruTip协议的也使先进的用户整合上游或downstream自动化流程与TruTip。例如的汉密尔顿easyBlood解决方案以分级分离的全血，也能与的的自动化TruTip提取方法，这会显着简化生物银行业务流程。如核酸定量，规范化，，PCR设置的，或DNA测序后提取工艺也很容易地集成与TruTip较大的液体处理平台。

核酸纯化是必要的大多数分子诊断，研究使用，和生命科学的应用。出现了各种方法，因为对时间的苯酚/氯仿提取，其中有许多是在离液序列高的盐的存在下用核酸结合的基本原则基于。精简和自动化提取过程中一直通过使用各种珠膜为基础的格式，与自旋过滤器，磁珠及相关方法，主宰生命科学行业（见例子2-13）。尽管有效，但已知粒子和膜的局限性，当面对具有挑战性的临床矩阵。例如，细胞膜和珠列标准，有小孔径（因此，高背压），离心机或真空系统要处理的订单，需要某种类型的支持。膜的物理特性，和胎圈列结果显着的流体阻力，从而限制了不同的样品，可以有效地处理无堵塞的消耗品，和/或总的（输入），可以是单方向的流路，通过处理的样品体积。相反，磁性粒子必须分布在整个样品中，搅拌。均匀分布的磁性颗粒的溶液内的需要限制了总的输入样品的体积，可以处理最磁珠消耗品。临床样品中的属性（如粘度或复杂性），可导致低效的磁性粒子浓度的一侧的管或棒。此外，二氧化硅微粒，可以折断的珠子在提取过程中，失去了自己的磁化强度和污染最终样本。 的TruTip技术的开发是为了克服这些核酸的样品处理的约束和限制14。通过嵌入的多孔整料内枪头，降低流体背压流量控制，使通过真空（ 即吸管抽吸）。此功能可以大大简化，从复杂样品类型（ 图1）纯化核酸的提取工艺和仪表。定制的单块的几何形状和孔隙率，以减少堵塞，而单块的厚度提供足够的样品体积范围从1.0到5.0毫升的核酸结合能力。样品吸液和分配过程中的双向流动样品提取物和高效的核酸回收和洗脱结合巨石之间允许长期居留的时间，并要处理超过枪头本身的容量为使比较大的样本量。我们以前报道的发展和应用的的手动TruTip程序净化流感RNA的鼻咽样本使用单一或多通道清莱万年移液器15。等效提取效率之间获得了自动化的QIAcube工作站手动TruTip方法的在A型流感10 6个基因拷贝每毫升鼻咽分泌物。本研究的目的是要证明中等和高通量，自动化TruTip核酸纯化过程的鼻咽分泌物（NPA）和其他临床相关样本类型，使用中常见的临床参考实验室的液体处理机器人。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material &lt;/strong&gt;</td></tr><tr><td>TruTip Influenza Extraction Kit (EPM TruTips)</td><td><a href="http://www.akonni.com/" target="_blank"><img src="/img/new_window.png" /> Akonni Biosystems, Inc.</a></td><td>300-11120</td><td></td></tr><tr><td>95% Ethanol</td><td>Acros Organics/ThermoFisher Scientific</td><td>AC615110040</td><td></td></tr><tr><td>99% Acetone</td><td>Sigma-Aldrich</td><td>270725-4L</td><td></td></tr><tr><td>DEPC-treated water</td><td>Life Technologies</td><td>AM9906</td><td></td></tr><tr><td>Reagent Reservoir, 30 ml</td><td>Eppendorf</td><td>960050100</td><td></td></tr><tr><td>Deep well plate 96/2,000 &amp;mu;l</td><td>USA Scientific</td><td>30502302</td><td></td></tr><tr><td>epT.I.P.S. Motion Filtertips, 1,000 &amp;mu;l</td><td>Eppendorf</td><td>960050100</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>epMotion 5070 System</td><td>Eppendorf</td><td>5070 000.000</td><td></td></tr><tr><td>Dispensing tool TM1000-8</td><td>960001061</td><td></td><td></td></tr><tr><td>Reservoir rack</td><td>960002148</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 1. &lt;/strong&gt; Reagents and equipment for automated RNA extraction from NPA.</td></tr><tr><td colspan="4">&lt;strong&gt;Reagent/Material &lt;/strong&gt;</td></tr><tr><td>TruTip gDNA Blood Extraction Kit (Hamilton TruTips)</td><td><a href="http://www.akonni.com/" target="_blank"><img src="/img/new_window.png" /> Akonni Biosystems, Inc.</a></td><td>300-20341</td><td></td></tr><tr><td>95% ethanol</td><td>Acros Organics/ThermoFisher Scientific</td><td>AC615110040</td><td></td></tr><tr><td>Proteinase K</td><td>AMRESCO LLC</td><td>E195</td><td></td></tr><tr><td>1 ml Hamilton filtered CO-RE 96 tip rack</td><td>Hamilton Robotics, Inc.</td><td>235905</td><td></td></tr><tr><td>1 ml Hamilton non-filtered CO-RE 96 tip rack</td><td>Hamilton Robotics, Inc.</td><td>235904</td><td></td></tr><tr><td>50 ml Reagent Trough</td><td>Hamilton Robotics, Inc.</td><td>187297</td><td></td></tr><tr><td>Deep Well 2 ml plate</td><td>USA Scientific</td><td>1896-2800</td><td></td></tr><tr><td>Nunc 96 DWP-2 ml</td><td>Thermofisher</td><td>27874</td><td></td></tr><tr><td>Reagent Trough</td><td>Fisher</td><td>14-222-412</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Hamilton STAR System</td><td>Hamilton Robotics, Inc.</td><td>173027</td><td></td></tr><tr><td>1 ml Independent Pipette Channels / Modular Arm</td><td>Hamilton Robotics, Inc.</td><td>173081/173050</td><td></td></tr><tr><td>1 ml 96-channel head</td><td>Hamilton Robotics, Inc.</td><td>199090</td><td></td></tr><tr><td>Tip Carriers</td><td>Hamilton Robotics, Inc.</td><td>182085</td><td></td></tr><tr><td>Sample Carriers/Inserts</td><td>Hamilton Robotics, Inc.</td><td>173400/182238</td><td></td></tr><tr><td>Plate Carriers</td><td>Hamilton Robotics, Inc.</td><td>182090</td><td></td></tr><tr><td>Multiflex Carrier</td><td>Hamilton Robotics, Inc.</td><td>188039</td><td></td></tr><tr><td>HHS2 Heater Shaker Unit</td><td>Hamilton Robotics, Inc.</td><td>199033</td><td></td></tr><tr><td>Rack Carrier</td><td>Hamilton Robotics, Inc.</td><td>188047</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 2. &lt;/strong&gt; Regents and equipment for 96-well genomic DNA extraction from whole blood.</td></tr><tr><td colspan="4">&lt;strong&gt;Reagent/Material &lt;/strong&gt;</td></tr><tr><td>TruTip R+D Circulating DNA Extraction Kit (Hamilton TruTips)</td><td><a href="http://www.akonni.com/" target="_blank"><img src="/img/new_window.png" /> Akonni Biosystems, Inc.</a></td><td>Call to inquire</td><td></td></tr><tr><td>100% ethanol</td><td>Sigma-Aldrich</td><td>459828-1L</td><td></td></tr><tr><td>Isopropanol</td><td>Acros Organics/ThermoFisher Scientific</td><td>AC327270010</td><td></td></tr><tr><td>Proteinase K</td><td>AMRESCO LLC</td><td>E195</td><td></td></tr><tr><td>Filtered 4 ml Tips</td><td>Hamilton Robotics, Inc.</td><td>184022</td><td></td></tr><tr><td>Unfiltered 1 ml Tips</td><td>Hamilton Robotics, Inc.</td><td>235939</td><td></td></tr><tr><td>96-Deep Well Plates</td><td>USA Scientific</td><td>1896-2800</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>Corning/ThermoFisher Scientific</td><td>05-526B</td><td></td></tr><tr><td>50 ml Reagent Troughs</td><td>Hamilton Robotics, Inc.</td><td>187297</td><td></td></tr><tr><td>120 ml Reagent Troughs</td><td>Hamilton Robotics, Inc.</td><td>182703</td><td></td></tr><tr><td>Large Volume 96-Pos Reagent Troughs</td><td>ThermoFisher Scientific</td><td>14-222-412</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>STARplus Autoload Workstation Base / Deck Module</td><td>Hamilton Robotics, Inc.</td><td>173025/190012</td><td></td></tr><tr><td>1 ml Independent Pipette Channels / Arm</td><td>Hamilton Robotics, Inc.</td><td>173081/173052</td><td></td></tr><tr><td>5 ml Independent Channel / Modular Arm</td><td>Hamilton Robotics, Inc.</td><td>184090/173050</td><td></td></tr><tr><td>Plate Carriers</td><td>Hamilton Robotics, Inc.</td><td>182090</td><td></td></tr><tr><td>Multiflex Carrier</td><td>Hamilton Robotics, Inc.</td><td>188039</td><td></td></tr><tr><td>Rack Carrier for 50 ml Reagent Troughs</td><td>Hamilton Robotics, Inc.</td><td>188047</td><td></td></tr><tr><td>120 ml Reagent trough carrier</td><td>Hamilton Robotics, Inc.</td><td>185290</td><td></td></tr><tr><td>Tip Carriers</td><td>Hamilton Robotics, Inc.</td><td>182085</td><td></td></tr><tr><td>50 ml Tube Carriers</td><td>Hamilton Robotics, Inc.</td><td>182245</td><td></td></tr><tr><td>24 Position Sample Carriers</td><td>Hamilton Robotics, Inc.</td><td>173400</td><td></td></tr><tr><td>32 Position Sample Carrier</td><td>Hamilton Robotics, Inc.</td><td>173410</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 3. &lt;/strong&gt; Reagents and equipment for large volume DNA extraction from plasma.</td></tr></tbody></table>

high-throughput, automated extraction of dna and rna from clinical samples using trutip technology on common liquid handling robots

TruTip是一个简单的核酸提取技术，利用该技术，一种多孔性，单片的连接基质的移液管尖端插入。的整体的几何形状可适应特定的移液吸头的体积范围从1.0到5.0毫升。孔隙度大的巨石使粘稠的或复杂的样品容易通过它以最小的流体背压。双向流量最大化整料和样品之间的停留时间，使内一个单一的TruTip的要处理大体积的样品。的基本步骤，不论样品的卷或TruTip几何形状，包括细胞裂解，核酸结合的内孔的TruTip整料，洗去未结合的样品组分和裂解缓冲液，和到适当的缓冲液洗脱，纯化和浓缩的核酸。属性和适应性表现在TruTip三个自动化临床样本处理协议，使用的Eppendorf epMotion 5070汉密尔顿STAR STARPLUS液体处理机器人，包括RNA分离从鼻咽分泌物，全血基因组DNA提取，提取胎儿DNA和母体血浆（分别）从大量富集。

实时PCR数据NPA流感RNA提取图5A所示。预计核酸提取效率是差不多的结果与手动版本的协议15。观察C T值平均为10 4 10 6的线性响应基因拷贝毫升-1修订流感（R 2 = 0.99和0.98的流感A和B，分别），标准偏差，平均C T值小于1周期。与的epMotion系统的交叉污染的情况下被显示在图5B中 ，，12阳性NPA样品中含有10 6个基因拷贝毫升-1 NPA穿插12缓冲液的空白。为阳性对照组的平均C T为30.16±0.14，和所有的缓冲空白阴性。总样本处理时间为16，28和40分钟分别为8,16和24个样品，。因为一个典型的临床鼻咽分泌物或棉签将含有10 7个基因拷贝毫升-1（假设每1000病毒颗粒TCID 50 16&gt; 10 4 TCID 50毫升-1流感17），自动化epMotion协议预计可有效的多数临床NPA标本。 在人基因组D​​NA上进行的分子检测的范围内，从全血中提取核酸的主要目标是产生无污染物的，高分子量的基因组DNA。 96个样品的自动化协议完成后在1小时之内，这是一种进步超过其它自动化系统（ 如 Promega公司马格尼西= 90分钟和Qiagen公司的QIAamp DNA血液BioRobot MDx系统进行= 2.5小时）。 图6A示出的紫外/可见光吸光度的档案同时处理与45试剂空白汉密尔顿STAR协议，与平均45阳性的血液样本<sub&gt; 260/280的比例为1.96，平均260/230比值为1.93。一个A 260/280比值介于1.7-2.0和A 260/230比值&gt; 1.7普遍反映非常纯净的DNA，无的残留盐，蛋白质或溶剂，和可接受的为最下游分子应用。的1％琼脂糖凝胶在图6B中示出所得到的基因组DNA为高分子量（&gt; 24 kb的），用最小的剪切。平均的全套45个阳性样品核酸产量为5.26±0.46微克每200微升全血，人类的DNA基于生命技术Quantifiler的人类DNA定量检测试剂盒。 图5B中所示的相同的交叉污染的研究进行穿插阳性样品平行萃取，与阴性对照缓冲液空白，所有的空格，再次通过PCR阴性（未示出）。还适用于许多其他的基于PCR的分析将纯化的基因组DNA（图中未示出）。 实时汇集母体血浆样品的的大体积TruTip程序处理的8个复制样品，结果示于图7。完成完整的协议（包括离线蛋白酶K孵化）约2.5小时，类似Qiagen公司的手动循环核酸试剂盒（没有类似的自动提取试剂盒尚未提供）。在所有复制的平均C T值是34.58±0.66和29.76±0.50，男性胎儿（CHY）和总DNA（CH1），分别表现出优异的可重复性的自动提取方法。在总DNA池的胎儿DNA（基因组当量）的浓度，计算标准的契合点分析比较的基础上，产生的平均％的胎儿DNA在所有样本的2.8％。实际％的胎儿DNA样品是未知的，因为前进行提取样品汇集。胎儿DNA组成非汇集血浆样品提取使用TruTip方法通常是高1.5倍，用Qiagen公司的循环核酸试剂盒（未示出）的结果比较。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig1highres.jpg" src="/files/ftp_upload/50356/50356fig1.jpg" /> 图1。的TruTip提取过程和工作流，而不管其他的样品制备或液体处理步骤的液体处理系统。自动化例程，这取决于具体的液体处理仪器和软件的能力可以被纳入。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig2highres.jpg" src="/files/ftp_upload/50356/50356fig2.jpg" /> 图2。 （A）的Eppendorf epMotion 5070样品板布局。 （B）安排的试剂/耗材绩优工作台样品板可以配置多达24个样品（1，5和9，分别列），虽然epMotion只能处理最多8个样品同时<a href="https://www.jove.com/files/ftp_upload/50356/50356fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig3highres.jpg" src="/files/ftp_upload/50356/50356fig3.jpg" /> 图3。汉密尔顿STAR甲板布局从全血的纯化基因组DNA（不按比例）。甲板仓1 =哈密尔顿1毫升过滤的提示; 2 =汉密尔顿1毫升非滤波的提示; = Akonni /汉密尔顿1毫升LPT 2毫米TruTips; =输入血液样本载体（血液收集管或离心管）; 5-9 = 290毫升试剂槽裂解缓冲液F，乙醇，洗涤缓冲J，洗涤液K表和洗脱缓冲A2的，分别为10 = 96 -EEP以及结合板; 11 = 96深井洗净J; 12 = 96深井K; 13 = 96深井洗脱板; 14 = HHS2汉密尔顿加热器/摇床Nunc公司96深井孵化板; 15 = 50毫升试剂槽洗净含有蛋白酶K <a href="https://www.jove.com/files/ftp_upload/50356/50356fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig4highres.jpg" src="/files/ftp_upload/50356/50356fig4.jpg" /> 图4。汉密尔顿STARPLUS甲板布局从大体积的血浆样品（不按比例）的纯化DNA，该系统配有8×5毫升通道和8×1毫升通道（未示出在甲板上的布局）。甲板位置1 =汉密尔顿4毫升过滤提示; 2 = Akonni /汉密尔顿5毫升TruTips; 3 =源血浆样品; 4 = 50毫升锥形管; 5 = 120毫升试剂槽含CN-CN-W1，W2和CN-W4 reageNTS; 6 =低容量的试剂槽含蛋白酶K CN-B2，CN-B3，EBA2，低潮和CN-W3试剂; 7 = 290毫升试剂槽含CN-L1的试剂; 8 = 290毫升试剂槽含CN 9 = 96深孔板的第1步; 10 = 96孔板步骤2 11 =样品纯化，最终产品的载体; 12 =汉密尔顿1毫升过滤的提示; 13 = Akonni /哈密尔顿1毫升LPT-B1试剂; ，4毫米TruTips。 <a href="https://www.jove.com/files/ftp_upload/50356/50356fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig5highres.jpg" src="/files/ftp_upload/50356/50356fig5.jpg" /> 图5。 （A）。从的自动化TruTip提取流感病毒实时荧光定量PCR结果添加到鼻咽部分泌物（NPA）。输入NPA量= 100微升，洗脱体积= 50微升。结果是平均3复制êxtractions从5个不同的NPA背景组（n = 15）每稀释水平和流感的目标。在LightCycler 480系统的qPCR实验条件先前所述15（B）无交叉污染时，检测到12个正面NPA样品穿插无模板对照和自动提取过程。 <a href="https://www.jove.com/files/ftp_upload/50356/50356fig5large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig6highres.jpg" src="/files/ftp_upload/50356/50356fig6.jpg" /> 图6。从全血中提取人类基因组DNA的结果。）紫外可见痕迹的NanoDrop 1000（赛默飞世尔）从来自10个随机选取复制B）1％琼脂糖凝胶的TruTip纯化的基因组DNA。 M =费舍尔24 KB最大DNA梯。泳道1 - 4 =〜100纳克纯化的基因组DNA从四个随机选择复制。 <a href="https://www.jove.com/files/ftp_upload/50356/50356fig6large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图7" fo:content-width="6in" fo:src="/files/ftp_upload/50356/50356fig7highres.jpg" src="/files/ftp_upload/50356/50356fig7.jpg" /> 图7。实时荧光定量PCR结果从八个复制TruTip自由流通从血浆中的DNA提取。记星号（*或**）的样品中提取在不同的日子。 CHY量化男性胎儿DNA和CH1量化总DNA存在（胎儿和产妇）。定量PCR上的LightCycler 480系统（罗氏）与先前公布的实验针对CHY和CH1 18。

Watch this Scientific Journal Video about High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots at JoVE.com

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip是一个简单的核酸提取技术，利用该技术，一种多孔性，单片的连接基质的移液管尖端插入。因此，样品制备格式是兼容的液体处理工具，可用于许多介质中，以高通量临床应用和样品类型。

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

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34.4K 次观看.  Akonni Biosystems, Inc.. TruTip是一个简单的核酸提取技术，利用该技术，一种多孔性，单片的连接基质的移液管尖端插入。因此，样品制备格式是兼容的液体处理工具，可用于许多介质中，以高通量临床应用和样品类型。

视频: High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

生物工程

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

生物化学

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

生物学

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, which can take up significant person-hours of bench labor and present a source of batch-to-batch variability. Lab automation with pipetting robots can reduce manual work, maximize throughput, and increase research reproducibility. Still, the steep starting prices of standard automation stations make them unaffordable for many academic laboratories. This article describes a proteomics sample preparation workflow using an affordable, open-source automation system (The Opentrons OT-2), including instructions for setting up semi-automated protein reduction, alkylation, digestion, and clean-up steps; as well as accompanying open-source Python scripts to program the OT-2 system through its application programming interface.

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

Shotgun 蛋白质组学 样品处理 由开源实验室机器人自动执行

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

发育生物学

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

摘要

探索更多视频

此视频中的章节

新一代测序为可操作的突变在固体和液体瘤检测

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

患者生物流体中肿瘤相关循环DNA的检测与监测

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

人死后脑组织中的单细胞群获得高品质的RNA

模块化 DNA 设备的自动机器人液体处理组件

Shotgun 蛋白质组学样品处理由开源实验室机器人自动执行

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

摘要

探索更多视频

此视频中的章节

新一代测序为可操作的突变在固体和液体瘤检测

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

患者生物流体中肿瘤相关循环DNA的检测与监测

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

人死后脑组织中的单细胞群获得高品质的RNA

模块化 DNA 设备的自动机器人液体处理组件

Shotgun 蛋白质组学 样品处理 由开源实验室机器人自动执行

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

Shotgun 蛋白质组学样品处理由开源实验室机器人自动执行