We thank Stephen Blazie, Karen Anderson, Josh LaBaer for advice and discussion. Karen Anderson, John Chaput, and Josh LaBaer for sharing reagents and instrumentation, Michael Gaskin and Andrea Throop for technical advise and protocols. Justin Wolter is a Maher scholar and thanks the Maher family for their generous support.

<html> 1.细胞培养（24-48小时之前转染） <ol><li> 24-48小时在转染前种子基于96孔平板的个数HEK293T细胞的足量转染。 注：对于一致转染中，板的细胞在足够的密度以有利于快速分裂，但不超过70-90％汇合在转染时。 </li><li>每个96孔板需要9×10 6个细胞（每孔75000细胞，并每板120的孔占使用贮存器和多道移液器的）。计算HEK293细胞（通常为〜20小时）的倍增时间，和种子细胞的适当数量的在转染时，以获得至少9×10 6个细胞。 A 145 mm圆柱培养板通常是足够3 96孔转染，当发展到〜90％汇合，剩余补种一个新的板块细胞〜10％。 </li></ol> 2.准备在转染_content“&gt;注意：应该在天转染前进行在步骤2.0-2.2的缓冲液和质粒DNA的制备，因为这些试剂的制剂可以是耗时的。 <ol><li>都可以通过一个公共的质粒库的30个人的3&#39;UTR克隆是与3&#39;LIFE测定兼容。手动或使用液体处理机器人纯化DNA质粒。用转染级碱裂解小量制备96孔套件，并按照制造商的说明。悬浮纯化向量〜100毫微克/微升每孔。 注：没有足够的荧光素酶信号将导致如果质粒浓度低于40毫微克/微升。 </li><li>获得使用图1B 24管道中的pLIFE-miRNA的载体30或克隆。重悬的载体，在500毫微克/微升的每个miRNA与空白对照质粒的浓度。 </li><li>由于的核转缓冲条件的敏感性，确保染材料（包括细胞和质粒）的总体积不会在96孔转染板的每个孔超过总液体的10％。为了实现这一点，浓缩pLIFE体miRNA质粒库存至少500毫微克/微升的浓度。 </li><li>制备10倍萤火虫萤光素酶缓冲液的试剂（ 见表1），和1×Renilla荧光素酶缓冲试剂（ 表2），其可存储长达6个月。 注：在DTT萤火虫荧光素酶缓冲区必须要在单次使用分装-20℃保存在溶液中。 </li></ol> 3.准备下列项目紧接转<ol><li>制备含有PBS的转染缓冲液，1.5％的HEPES，pH值7.0。准备这个新鲜的，虽然它可能会在4℃下保存长达1个月而不染效率明显下降。在制定和缓冲区质粒DNA卷，承担120反应为每个96孔板苏fficiently占的移液量和使用液体水库和多道移液器丢失的错误。分装，每孔转缓冲区18微升（120孔/ 96孔板=每盘2.16毫升），并预留。 注：细胞电穿孔装置是用于转染细胞中的缓冲液条件极其敏感。当精度准备缓冲区将确保设备的稳定性能。执行检测，以防止缓冲器的蒸发，特别是通过减少该缓冲器是左露出在微量离心管中，96孔板，水库和电极板的时候，额外必须小心。 </li><li>储备四口井以下控制，无pLIFE-3&#39;UTR（测量荧光素酶测定的背景），pLIFE-SV40 3&#39;UTR（负目标控制），阳性对照的miRNA＃1，阳性对照的miRNA＃2。 注：这些载体公开提供30。另外，任何先前验证目标可以用作阳性对照。 </li><li>暖媒体，胰蛋白酶（0.25％）至37℃。 </li><li>到96孔细胞培养板各孔中，加入200微升DMEM补充有10％FBS，1％青霉素/链霉素，并放置在转染后37℃培养箱中使用。 </li><li>打开所有细胞电设备之后的配套软件。采用脉冲代码为FF120 HEK293T细胞和PBS / HEPES缓冲液。 </li></ol> 4.制备质粒DNA和细胞混合物注意：下面的协议假定转染三个96孔平板在一个实验中对于具有两个微RNA（miRNA-＃1和miRNA-＃2）的画面。每块板将对应于相同的96孔平板的pLIFE-3&#39;UTR质粒，并进行处理三次pLIFE-miRNA的空白，pLIFE-miRNA-＃1，或pLIFE-miRNA-＃2。 <ol><li>准备3股pLIFE-miRNA的转染+缓冲每个miRNA。此库存应占每个总体积的50％（10微升）好了，再乘以120口井。因此，每个股票应该包含1.08毫升缓冲区+ 120微升质粒DNA（pLIFE-miRNA）的。 </li><li>通过洗脱介质，用PBS轻轻洗涤，并用〜5毫升0.25％胰蛋白酶，在37℃处理5分钟，除去自145 100mm培养板上的细胞。中和胰蛋白酶用等体积的介质，并沉淀细胞，在300×g离心5分钟。 </li><li> 5-10毫升媒体（视细胞密度和细胞计数准确范围）中删除〜胰蛋白酶/媒体和重新悬浮颗粒。 </li><li>计数使用细胞计数器细胞。确保细胞&gt; 95％存活，并在机器的准确范围。 注：不准确的细胞计数可导致非常高的细胞浓度（&gt; 6.0×10 6 /毫升）。转染太多细胞可显着降低的miRNA的效率，减少质粒靶向：细胞比率和/或降低转染效率。 </li><li>等分三个管各含9×10 6个细胞中，对应于细胞řequired一个96孔板的转染。旋转细胞在300×g离心3分钟。 </li><li>取出介质。一定要删除尽可能多的媒体尽可能与颗粒的最小干扰为多余的介质会影响转染效率。 </li><li>悬浮细胞1.2毫升转缓冲/ miRNA的质粒的混合物，并预留。 </li><li>以下步骤pLIFE-3&#39;UTR质粒转染缓冲细节悬浮。由于这发生在96孔板，注意避免缓冲区蒸发通过覆盖板在所有时间。 <ol><li>使用多通道移液管，将32.4微升转染缓冲液到96孔PCR板的每个孔（9微升[每转] * 3 [板材] * 1.2 [占吸管错误]）。 </li><li>加入3.6微升迷你坦然pLIFE-3&#39;UTR质粒（〜100纳克/微升），以每口井调匀。 </li><li>移取10微升该混合物到96孔转染板和盖的每个孔中。 </li></ol></li></ol><ol><li>移动1.2毫升第一个单元格/缓冲/ pLIFE-miRNA的质粒混合物进入水库。拌匀。 </li><li>加入10微升这种混合物进入已含有10微升转染缓冲/ pLIFE-3&#39;UTR第96孔转板。移液器向上和向下几次拌匀。 注：细胞在缓冲的等悬浮将确保甚至和电流的通过试管彻底通道和最大化转染效率。 </li><li>将96孔转板细胞电设备上，并开始转染。 </li><li>一旦转染完成后，从96孔培养板中添加100微升预热媒体向96孔转染板的每个孔中并充分混合。移动100微升每孔放入96孔培养板。 </li><li>混合的细胞在培养板吸移管垂直放置在井的中心，因为细胞将趋向于聚集在瓦特的侧面ELL除非适当地混合。 </li><li>对其余的两个板重复5.1-5.5。 </li><li>清洁96孔转板<ol><li> 96孔转染板可以回收洗涤用70％乙醇，以确保没有结转的实验之间的核酸。执行使用喷雾瓶完全填充每个孔，接着擦拭过量乙醇上的电极条带（底部侧），并允许在培养罩的转板完全干燥两个70％乙醇洗涤。 注意：我们已通过转染的12个孔用2微克pmaxGFP质粒每个入HEK293T细胞测试结转DNA污染，接着用70％EtOH中的单次洗涤，并用无质粒DNA中的第二次转染。有了这个极高浓度的质粒，极其明亮记者，和单一的洗，有任何的重复12转染的没有观察到荧光。 </li></ol></li><li>培养细胞48-72小时，在37℃，随后双重荧光素酶擦除检测。 </li></ol> 6.细胞裂解液准备萤光素酶检测<ol><li>稀释裂解缓冲液与4份水，1份5倍于一个储存器被动裂解缓冲液。计算26微升/孔，加入约20多卷，占在水库损失。 注：缓冲储存在-20ºC并且可以以允许5×缓冲接近室温将提高注精度非常粘稠，因此之前。 </li><li>每口井用荧光显微镜转染效率分析。注水井中的任何不一致未转染效率（&gt; 90％的转染效率），或者是表达RFP表示超员或媒体疲惫的低水平。从分析中删除这些井。 </li><li>从细胞中完全删除媒体，小心不要洗脱过快，这将导致细胞分离。 注：剩余媒体会稀释跨越experime值和裂解波动原因NTS。 </li><li>加入26微升的裂解液以每口井，并放置在板振动筛/摇杆在低/中转速〜20-30分钟。利用这段时间准备萤光素酶缓冲液，洗净和总理光度计（S），并转移裂解液不透明测量板。 </li></ol> 7.双萤光素酶检测注：如果多个板块被测量顺序在一个光度计，创建缓冲区预混的一切，除了ATP和底物，加入这些试剂然后调节pH值应立即用每块板后再使用。 ATP和底物可能随时间降解;在时间这些试剂是在缓冲器量的一致性将改善在多个板的一致性。 <ol><li>制备1×萤光素酶缓冲液（ 见表1）： <ol><li>裹两管（通常15/50毫升离心管）含萤火虫和海肾缓冲器，铝箔，作为底物可能是光敏感。 </li><li>准备1X萤火虫荧光素酶的缓冲。加入1毫升每五个10×萤火虫萤光素酶试剂，加入EGTA最后，到5ml H 2 O至最终1倍浓度。 <ol><li>添加0.025克ATP至10ml 1X萤火虫缓冲。颠倒几次混合。保持的ATP在冰上在任何时候。 ATP会降低，因此，如果测量多个板块顺序，缓冲区必须为每个附加板这一步作出新的开始。 </li><li>加入100微升100甲虫萤光素（底）（ 表1）缓冲应该改变的基础上pH值黄色。 </li></ol></li><li> 1×Renilla荧光素酶缓冲液重建：每96孔板，等份加入10ml 1“ 的Renilla缓冲”。 <ol><li>加入100微升BSA（44毫克/毫升的股票）。 </li><li>如果筛查多个板块，单独预混成10毫升分装。 </li><li>加入100微升腔肠素中缓冲的（预先等分并储存在100倍浓度） </li></ol></li> &lt;李&gt;调整pH值的1x萤火虫缓冲至8.0，随后1倍的Renilla缓冲至5.0用NaOH和HCl。 注意：每个缓冲器的活性，和海肾缓冲区以猝灭萤火虫萤光素酶活性的能力高度依赖于pH值。为一致的结果是非常精确的在此步骤。 </li><li>带来每个缓冲区（对应于1 96孔板）的体积至10.5 ml至适应光度计灌注。 </li></ol></li><li>转移裂解物至白色不透明的板：此时的细胞应在为〜20分钟的裂解缓冲液。通过各个使用多道移液器以及25微升，一定要吸管上下彻底打破了细胞团块和均质化的裂解液。 </li><li>准备光度计。打开光度计和选择协议在DLR文件夹，名为“DLR具有两次喷射”。其他格式不与数据分析管道（下面）兼容。 <ol><li>选择井为t相关捐资（所有的孔是默认值）。 </li><li>扩展&#39;延迟测量之前“设置为5秒，用10秒的测量时间（见29为解释）。 </li><li>毛细管洗涤步骤：水3倍，3倍乙醇，水3倍，3倍干燥。总理缓冲区一旦进入垃圾，然后黄金第二次回缓冲管，以确保混合。第一和素它注入萤火虫缓冲器中的左毛细管，随后海鳃在右毛细管。 </li></ol></li><li>引发荧光素酶测定法。每板应采取〜48分钟阅读。完成后保存文件，然后再重复洗涤步骤，并关闭光度计。 注意：多个板可读取并存储在相同的数据excel文件，然而问题可以与多个板遇到读取其中光度计程序会崩溃。一定要保存所有的数据测量之间并采取截图，如果程序崩溃之前保存是可能的。 <ol><li>更换旧的缓冲区新的，是一定要贷至少两次与新的缓冲区开始新的板块之前。 </li></ol></li></ol> 8.数据分析<ol><li>利用Excel表格“3&#39;LIFE -单盘分析”和“3&#39;LIFE -多片分析”，可从<a href="http://www.mangonelab.com">www.mangonelab.com</a> 。原始数据从光度计输出文件萤火虫和Renilla荧光素酶测定复制到对应于所述负状态，miRNA的＃1和miRNA＃2进入“3&#39;LIFE -单平板分析”位置的电子表格。 </li><li>该电子表格会自动计算萤火虫/ 海肾比和规范每个miRNA到相应的阴性对照，并在每个板压制正常化值。 注：此表会自动识别井低荧光素酶信号，彰显显著压抑的水井，并提供repressi措施上在整个板。进行统计分析的详细解释见24。 <ol><li>对应于复制＃1测试每个miRNA的位置 - “多片分析3&#39;LIFE”电子表格从“归RI指数”框中的值复制，到相应的单元格。重复在不同的日子进行的所有生物复制。 </li></ol></li><li>如果需要的话，插入在列B和D中的基因名称和目标预测状态，分别。 <ol><li>允许电子表格来自动计算所有板的平均值。观察在96孔格式作为热图（ 图2）中的数据。该文件还排在列表格式的数据（ 图2）。 注：压制索引是用于识别推定的miRNA靶的量度。不同严格性参数都可以使用，根据个人喜好。平均而言，我们认为低于0压制指数可能命中。图8和用t检验（P -值&lt;0.05）统计学显著。 如图基于这些标准3的潜在目标将以红色突出显示。 </li></ol></li></ol>

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W., Hannon, G. J., Darnell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+mode+of+microRNA+target+recognition.">An alternative mode of microRNA target recognition.</a> Nat Struct Mol Biol. 19 (3), 321-327 (1038).</li><li>Jiao, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+targeting+oncogenes+are+down-regulated+in+pancreatic+malignant+transformation+from+benign+tumors.">MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors.</a> PLoS One. 7 (2), e32068 (2012).</li><li>Wolter, J. M., Kotagama, K., Pierre-Bez, A. 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This work is supported by funds from the College of Liberal Arts and Science and the Biodesign Institute at Arizona State University and NIH Exploratory/Developmental Research Grant 1R21CA179144-01A1.

所述3&#39;LIFE测定标识在高通量3&#39;UTRs官能miRNA靶。此法对研究者谁希望通过实验找出了大量的假定目标他们感兴趣的miRNA的有用。该3&#39;LIFE测定法是有效的方法，以查询3&#39;UTR从动调节，在该测定法提供了一个功能度量的miRNA靶向的，以及一个单一的记者的二进制测试:: 3&#39;UTR针对单一的miRNA可以自信地解决各个基因的靶向状态。为了验证这种方法，我们筛选了275 3&#39;UTRs一个面板和针对两种miRNA?...

该方法的总体目标是检测并精确地映射微小RNA（miRNA）的目标高通量。 miRNA是内源性非编码RNA〜22个核苷酸长度。以下转录和加工，成熟miRNA在蛋白质复合物掺入称为诱导的沉默复合物（RISC）的RNA。每个miRNA引导RISC靶向主要位于信使RNA的3&#39;非翻译区（3&#39;UTRs）（的mRNA）分子，从而在任一翻译抑制或mRNA切割1。 miRNA的认识到基于标准的Watson-Crick和G靶位点：U摆动碱基配对，并在退化性质，包含多个错配碱基对和凸出的区域。许多miRNA的大致从植物保守到人类2,3，在那里他们发挥生物学作用的多元化。在后生动物miRNA能够影响多个生物过程，包括细胞命运决定4，发育时间5 </sup&gt;，并经常表现出组织特异性表达模式6,7。 miRNA的异常表达也可以导致异常的基因调控，其可以具有关于只对靶基因的功能基于细胞行为实质性影响。这样，微RNA被链接到一个宽范围的疾病，包括神经变性8,9，糖尿病10和癌症11。生物信息学和湿式工作台的方法表明，每个miRNA可以是能够靶向数百至数千不同的mRNA 12-14，表明高通量或全基因组的方法是必需的，以探测该大潜在的相互作用的池。 鉴定的靶基因是机械地限定的miRNA功能的重要组成部分，并且这样做的研究人员必须能够揭示大规模目标。几种方法已经被开发，以确定miRNA靶，包括生物信息学预测算法，高通量测序有针对性的mRNA，并根据记者分析。每一种方法都有固有的长处和短处。给出了miRNA的定位是通过序列特异性的指导下，最显着的核苷酸与miRNA的2-6的（称为点火区），若干算法已经被开发，以在整个许多生物的基因组中预测的miRNA靶。这些算法使用证实的miRNA靶的观察到的碱基配对图案培训，并经常利用的参数，如严格种子配对，现场保护，和/或热力学稳定性15。而这些滤波器缩小大量具有足够互补性的推定的目标，以仅高置信指标，它们可以排除种间特异性和非规范miRNA靶位点，其中最近的证据表明有广泛16-24。此外，这些预测没有考虑到mRNA加工的行为排除miRNA靶位点，如替代多聚腺苷酸账户机制25，RNA编辑26，RNA甲基化27，并协同结合。因此，较高的假阳性和假阴性率已经报道了很多算法22,24,28。虽然这些算法是有用的，以确定用于随后的实验验证候选miRNA靶，这些高错误率限制的生物信息学方法进行系统的miRNA靶标检测的功效。 为了系统地探测和给定的miRNA之间的相互作用可能有针对性3&#39;UTRs我们已经开发了一个名为发光标识功能元素在3&#39;UTRs（3&#39;LIFE）24高通量检测。此测定法测量的直接相互作用，并测试3&#39;UTR的通过使用双荧光素酶报告系统的查询的miRNA翻译抑制。在这个系统中，感兴趣的基因的3&#39;UTR克隆萤火虫荧光素酶（fluc）记者阅读框架的下游。记者利弊truct被共转染在HEK293T细胞中的查询的miRNA。 miRNA的定位是通过测量测试fluc :: 3&#39;UTR记者和一个第二非特异性海肾萤光素酶报道之间的相对变化来确定。重要的是，荧光素酶检测检测影响记者的平移功能输出的miRNA / mRNA的相互作用。这是优于传统方法的关键优势检测miRNA调节，如RT-qPCR的和Western印迹，在这绕过在mRNA降解和翻译抑制的差异，以及在蛋白质丰度的独立的3&#39;UTR基于调节的变化。 萤光素酶测定法被广泛应用，以验证由于其相对简单和灵敏度，但它们在高通量筛选使用直接miRNA靶是通过用消耗品的试剂相关的高费用的限制，缺乏从公共来源3&#39;UTR库，以及没有标准化的荧光素酶PROTocols，导致难以在多个数据集进行比较的功能抑制。为了方便使用3&#39;LIFE分析，我们已经将重点放在了实验设计，利用非商业转24和 ​​荧光素酶试剂29的简化，创造了3&#39;UTR库定期更新和扩展，并且可通过公共质粒库30。 在3&#39;LIFE检测的可扩展性使大3&#39;UTR库筛选而不向偏置确定生物信息学的基因屏幕由给定miRNA的靶。除了测试规范和预测的相互作用，这种系统的方法允许通过非规范和/或物种特异性相互作用驱动的新靶标鉴定。重要的是，miRNA的对蛋白生产靶向的影响一般理解为导致适度翻译抑制15,31 &lt;/ SUP&gt;，表明miRNA调节的一个主要作用是微调蛋白输出，防止基因表达的异常水平，并提供鲁棒性与细胞的具体方案32,33。萤光素酶测定法结合固有大量在3&#39;LIFE屏幕负的miRNA / mRNA的相互作用的灵敏度允许检测的miRNA的靶上的大量基因的微妙的影响，和基因网络的多个组件的识别那由给定的miRNA 24进行调节。 在这里，我们描述了3&#39;LIFE协议，并证明它的可行性通过筛选2良好表征的miRNA中，miR-10b和让-7c的对275人的3&#39;UTRs（ 图1）一个面板。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents</td>
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			<td height="21" style="height:21px;">glycylglycine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
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		<tr height="21">
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			<td style="width:119px;">A2392</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well miniprep plate</td>
			<td style="width:71px;">Pall</td>
			<td>8032</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well shuttle plates</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">V4SP-2096</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5x Lysis Buffer</td>
			<td style="width:71px;">Promega</td>
			<td>E1941&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
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			<td height="21" style="height:21px;width:216px;">Instruments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well GloMax Plate Reader</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">E9032</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biomech FX Liquid Handler Robot</td>
			<td style="width:71px;">Beckmann</td>
			<td style="width:119px;">A31842</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4D-Nucleofector Core Unit</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">AAF-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well Shuttle System</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">AAM-1001</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Counter Countess</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C10227</td>
		</tr>
	</tbody>
</table>

detection of mirna targets in high-throughput using the 3'life assay

功能元件发光鉴定3&#39;UTRs（3&#39;LIFE）允许数百查询3&#39;UTRs的一个阵列内快速识别特定的miRNA的目标。目标识别是基于双荧光素酶测定法，其检测在mRNA水平通过测量平移输出，给人的miRNA靶向功能的读出绑定。 3&#39;LIFE使用非专用的缓冲液和试剂，并公开提供记者库，使得全基因组屏幕可行和成本效益。 3&#39;LIFE可以执行无论是在标准实验室环境或扩大了使用的液体处理机器人和其他高通量检测仪器。我们示出了使用克隆在96孔板人类3&#39;UTRs和两个测试miRNA的数据集的方法， 让-7C和miR-10b中 。我们展示了如何执行DNA制备，转染，细胞培养和萤光素酶测定在96-孔格式，并提供工具用于数据分析。总之3&#39;LIFE是高度可重复的，快速的，系统的，并确定高可信度的目标。

光度计的输出文件包含原始测量为萤火虫和Renilla荧光素酶的蛋白质。这种原始的格式与“3&#39;LIFE -单盘分析”兼容，“3&#39;LIFE -多片分析”表格可从Mangone实验室网站（ <a href="http://www.mangonelab.com">www.mangonelab.com</a> ）。单板分析表格自动计算萤火虫/ 海肾比，标准化每个miRNA到相应的阴性对照，并在每个板压制标准化值。此电子表格自动识别井低Renilla荧光素酶信号，凸显井表现...

Watch this Scientific Journal Video about Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay at JoVE.com

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

detection-of-mirna-targets-in-high-throughput-using-the-3-life-assay

Research

JoVE Journal

Biology

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

10.0K 次观看.  Arizona State University. Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

视频: Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

科研

生物学

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...