Name: detection of mirna targets in high-throughput using the 3'life assay
Uploaded: 2015-05-25T13:00:00.000+00:00
Duration: 12 min 49 s
Description: Watch this Scientific Journal Video about Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay at JoVE.com

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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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><tbody><tr><td>Reagents</td><td></td><td></td><td></td></tr><tr><td>glycylglycine</td><td>Sigma</td><td>G1127-25G</td><td></td></tr><tr><td>Kx PO4</td><td>Sigma</td><td>P2222</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>FLAAS</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>MgSO4</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>CoA</td><td>Sigma</td><td>C4282</td><td></td></tr><tr><td>luciferin</td><td>Sigma</td><td>L9504</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Na2EDTA</td><td>Sigma</td><td>E0399</td><td></td></tr><tr><td>K H2 P O4</td><td>Sigma</td><td>1551139</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>NaN3</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>Coelenterazine</td><td>Sigma</td><td>C3230</td><td></td></tr><tr><td>PBS/HEPES</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>DMEM</td><td>Sigma</td><td>D5546</td><td></td></tr><tr><td>FBS</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>Pennicilin/Streptomicin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Trypsin</td><td></td><td>T2600000</td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>MaxiPrep Kit</td><td>Promega</td><td>A2392</td><td></td></tr><tr><td>96-well miniprep plate</td><td>Pall</td><td>8032</td><td></td></tr><tr><td>96-well shuttle plates</td><td>Lonza</td><td>V4SP-2096</td><td></td></tr><tr><td>5x Lysis Buffer</td><td>Promega</td><td>E1941&amp;nbsp;</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>96-well GloMax Plate Reader</td><td>Promega</td><td>E9032</td><td></td></tr><tr><td>Biomech FX Liquid Handler Robot</td><td>Beckmann</td><td>A31842</td><td></td></tr><tr><td>4D-Nucleofector Core Unit</td><td>Lonza</td><td>AAF-1001</td><td></td></tr><tr><td>96-well Shuttle System</td><td>Lonza</td><td>AAM-1001</td><td></td></tr><tr><td>Cell Counter Countess</td><td>Invitrogen</td><td>C10227</td><td></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，凸显井表现出抑?...

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'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

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 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 ...

科研

生物工程

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

遗传学

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

发育生物学

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

转移性结直肠癌患者循环微 rna 自定义面板的检测

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

癌症研究

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

生物化学

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

生物学

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

检测miRNA靶在高通量的使用3'LIFE分析

摘要

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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

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

转移性结直肠癌患者循环微 rna 自定义面板的检测

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

用于植物微RNA检测和定量的RNA斑点分析

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3'UTR记者构造的microRNA识别目标在贴壁细胞

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

检测miRNA靶在高通量的使用3'LIFE分析

摘要

探索更多视频

此视频中的章节

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

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

转移性结直肠癌患者循环微 rna 自定义面板的检测

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

用于植物微RNA检测和定量的RNA斑点分析

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3'UTR记者构造的microRNA识别目标在贴壁细胞

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

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