我们想感谢阿米里根仔细阅读的手稿。支持这项工作是由NGFN加授＃01GS0801，MRC奖学金授予G1002523 NHSBT授予WP11-05，LD和DFG授予FR2938/1-1 CCF

1。代谢标记4 thiouridine 实验装置的详细计划/调度， 例如 ，当添加4SU细胞培养和收获样品。计划在每个条件之间的至少5分钟。只有一个条件处理细胞的时间。处理最大。在一个给定的时间3 - 5个菜。尽可能快地处理细胞，以尽量减少在温度和CO 2水平的变化。避免明亮的光线后的细胞暴露4SU增加，因为这可能导致4SU标记的RNA的细胞蛋白的交联。 标签开始<ol><li>解冻4 thiouridine（4SU）之前使用和吸管4SU每个条件进入无菌猎鹰管所需的金额。 </li><li>以细胞培养液（5毫升每10厘米的培养皿）关闭菜肴所需金额，并添加含4SU猎鹰管调匀。取出并丢弃菜的剩余介质。 </li> &lt;LI&gt; 4SU含介质倒流的菜肴。 </li></ol>结束标记<ol start="4"><li>除去细胞的培养基从细胞中。加入5毫升Trizol法时，每盘。对于复杂的实验，其中包括多个时间点或条件，这一步最好由两个人，除去培养基，加入Trizol试剂和收获的溶胞产物。 </li><li>完整的细胞裂解在室温下孵育5分钟。 </li><li>使用10毫升吸管仔细冲洗板块，与增加的Trizol。这有助于完整的细胞裂解样品回收率。小心轻放的Trizol在与皮肤或眼睛接触时，是非常危险的！有苯酚烧伤手的解毒剂（ 如聚乙二醇300 400在工业甲基化酒精（70:30））。将样品转移到聚丙烯管中。请注意标准猎鹰管不抵抗这些高克部队）。样品可以储存在-20℃下至少1个月，直至总RNA中，我准备投入战斗。 </li></ol> 2。 RNA制备使用改良的Trizol协议<ol><li>加入1毫升氯仿（0.2毫升，每毫升Trizol法）和剧烈摇晃，持续15秒。在室温下孵育2 - 3分钟。 </li><li>在13000×g离心15分钟，在4℃下</li><li>水的上层相（含有RNA）转移到一个新的15毫升的聚丙烯管中。 </li><li>添加½两个RNA沉淀缓冲液和异丙醇的反应体积（ 例如，3毫升上清液加1.5毫升的RNA沉淀缓冲液和1.5ml异丙醇）。 </li><li>拌匀。在室温下孵育10分钟。 </li><li>在13000×g离心10分钟，在4℃下弃上清。 </li><li>后短暂离心（5000×g为30秒）和200微升移液器去除残留的异丙醇。 </li><li>加入等体积的75％乙醇中，并摇动试管，直到颗粒分离。避免打破它成很多小块，因为这可能使残留物清除的L乙醇困难。 </li><li>在13000×g离心10分钟，在4℃下弃上清。 </li><li>简要降速RNA并取出剩余的乙醇200μl的吸管。重复步骤和除去剩余的乙醇与20微升吸管。这两个步骤之后，没有进一步的干燥沉淀应该被执行。 </li><li>加入100μl每股H 2 O 100微克RNA产量预期，并拌匀上下吹打5 - 6次，以帮助溶解RNA。 </li><li>溶解和变性的RNA通过加热到65℃下进行10分钟（振动筛），并立即在冰上放置。 </li><li>测量RNA浓度使用NanoDrop分光光度计在260 nm处，按照制造商的说明。这种RNA可以储存在-80℃下至少1个月。 </li></ol> 3。巯基新转录RNA的特定的生物素化<ol><li>开始60 - 80微克的细胞总RNA。 </li><li>组成贴标反应。以下吸管为了（每微克RNA​​）： <ol class="lalpha"><li> 1微升10X生物素缓冲区</li><li> 7μlRNA提取（含1微克RNA稀释于无核酸酶的H 2 O） </li><li> 2微升（1毫克/毫升DMF）生物素-HPDP </li></ol></li></ol>随时添加的生物素HPDP的最后，并立即混合吹打。的情况下，析出物的生物素，二甲基甲酰胺的含量可以增加至终浓度为40％。 <ol start="3"><li>在室温下孵育1.5小时，用旋转。 </li><li>加入等体积的氯仿。大力混合。孵育2 - 3分钟，直到相开始分离，气泡开始消失。 </li><li>在20000×g离心5分钟，在4℃下小心转移到一个新的管中的上层水相。 </li><li>重复一次步骤3.4和3.5。您可能要执行此步骤2毫升锁相凝胶重型管，以减少损失的RNA。 </li><li> RNA沉淀：加入1/10体积的5 M氯化钠和等量的异丙醇到水相中。 </li><li>在20000×g离心20分钟，在4℃下弃上清。 </li><li>加入等体积的75％乙醇，离心，在20,000×g下在4℃下10分钟，弃去上清液。 </li><li>短暂离心，用200微升吸管，去除残留的乙醇。 </li><li>短暂离心，去除残留的乙醇与20微升吸管。 </li><li>不要让RNA干。重新暂停在50 - 100微升H 2 O（〜1微升，每1微克输入RNA）。拌匀上下吹打5 - 6倍。 </li><li>检查RNA质量通过electrophoretical分析的排除RNA降解。 </li></ol> 4。斑点杂交分析4SU成立（可选） 4SU​​掺入可以很容易地由点生物素标记的RNA印迹分析。这是一个可选的步骤，允许4SU的掺入率相对生物素标记的DNA寡控制的故障排除和估计。这个实验我们recommend使用碘代乙酰基-生物素，而不是生物素-HPDP 4SU标记的RNA在步骤3.2中的生物素化。这将导致在一个不可逆的生物素化4SU-RNA。因此，基于列的方法（ 例如 RNeasy试剂）可以用于恢复较少量的生物素化的RNA（ 例如，5微克）。虽然RNA的生物素化使用生物素-HPDP也适于该测定的，所得到的信号弱，信噪比逊于（ 图3）。 <ol><li>按照协议4SU细胞总RNA中所描述的第1和第2的标签和隔离。 </li><li> Biotinylate 4SU标记的RNA中所描述的更换生物素HPDP的第3与碘代素和执行两个氯仿抽提完全除去过量的碘代生物素残留。 </li><li>异丙醇/乙醇沉淀恢复生物素化的RNA或使用基于列的方法（ 例如 RNeasy试剂）的情况下，少量的RNA（&lt;10微克）被使用。 </li><li>孵育的泽塔膜在无核酸酶水10分钟摇动。 </li><li>无核酸酶水出膜，将膜之间两个干净的纸巾用力按压，除去过量的液体。空气中干燥5分钟的膜，将导致更好的点。 </li><li>对于每个样品，准备20μl的200纳克/μlRNA提取，用冰冷的斑点印迹结合缓冲液中（10mM氢氧化钠，1mM EDTA）中。应用5微升稀释（ 即 1微克RNA），以及三个连续稀释10倍（ 即 100，10，1毫微克RNA），通过移液的Zeta膜。通过一个空架移液器枪头可以提供均匀分布的间距。或者，使用斑点印迹装置，根据制造商的指示。 </li><li>中应用5μl的生物素标记的DNA寡核苷酸的浓度范围从20毫微克/微升至20皮克/微升（ 即 100将0.1 ng寡）作为阳性续ROL通过移液膜。使用生物素，4SU天真的样品作为阴性对照。 </li><li>空气中干燥5分钟的膜。 </li><li>孵育30分钟，在40ml封闭缓冲液中与摆动膜。 </li><li>温育膜15分钟1:1,000链霉抗生物素蛋白 - 辣根过氧化物酶（5毫升PBS + 5毫升20％SDS +加入10μl链霉抗生物素蛋白 - 辣根过氧化物酶与10毫升） </li><li>在40毫升PBS + 10％SDS（20毫升PBS + 20毫升20％SDS）中洗膜两次，持续5分钟。 </li><li>在40毫升PBS中洗膜两次+ 1％SDS（38毫升PBS和2毫升20％SDS），持续5分钟。 </li><li>洗膜两次，在40毫升PBS + 0.1％SDS中（40毫升PBS +加入200μl20％SDS），持续5分钟。 </li><li>除去过量的流体通过将膜之间两个干净的纸巾，并牢牢按住它们。 </li><li>可视化膜结合HRP使用ECL每制造商的指示。 </li><li>放置在塑料箔膜/袋，去除气泡和孵育2分钟，在黑暗。 </li><li>公开膜影片1 - 5分钟。 </li></ol> 5。标记和未标记的RNA链霉亲和素磁珠分离<ol><li>热清洗缓冲液（3毫升每个样品）至65℃的水浴中。 </li><li>准备新的100毫米二硫苏糖醇（DTT）在无核酸酶H 2 O做滗15 - 30毫克的DTT粉放入干净的50毫升猎鹰管上放置超精细的规模。称重，并添加所需金额无核酸H 2 O </li><li>热生物素化的RNA样品，以65℃下变性10分钟，并立即在冰上。 </li><li>地点列μMacs成磁性立场。我们建议不要处理时间超过12个样品（6 - 8个样品是最优的）。 </li><li>有预平衡美天旎列1毫升室温洗涤液。这将需要约15分钟。 </li><li>同时，加100微升50 - 100μl的生物素标记的RNA链霉亲和素珠。在室温下孵育15分钟，用旋转。 </li> &lt;<html> LI&gt;如果还没有发起任何列排水到现在，这可以促进用戴手套的手指轻轻按下顶部的列。一旦流量已经开始列容易流失。 </li><li>应用的RNA /有孔玻璃珠的列。丢弃的流量通过，除非你想收回未标记的RNA组分（见第7节）。 </li><li>洗涤三次，用0.9毫升的65℃下洗涤缓冲液（1毫升移液吸头加样缓冲液，在65℃时，收缩）。 </li><li> 0.9毫升室温下洗涤​​缓冲液洗三次。 </li><li>吸取700微升缓冲RLT（Qiagen公司的RNeasy MinElute清理套件，）进入新的2毫升管，并把它们列下方。 </li><li>新转录的RNA进入RLT缓冲液洗脱，通过添加100μl的100mM DTT的列。 </li><li>执行第二次洗脱轮3分钟后到同一管加入100微升100毫米的DTT。 </li></ol> 6。新转录的RNA恢复ontent“&gt;继续的RNeasy MinElute清理协议（QIAGEN）按照制造商的说明25无核酸酶H 2 O的测量RNA浓度洗脱使用NanoDrop分光光度计，为了避免提交前需要解冻和重新冻结RNA高通量的检测，我们推荐的紧接之后的新转录的RNA制备的cDNA的纯化。2.5微升新转录的RNA的在20微升cDNA的合成混合用于cDNA合成按照制造商的说明使用。执行实时定量RT-PCR法用1控制：10稀释的cDNA混合。专区RNA在-80℃下 7。恢复未标记的未绑定RNA（可选） 未结合的RNA的情况下，需要恢复的流量通过收集和组合（列后，加入RNA的链霉抗生物素蛋白微球溶液），进行后续沉淀的第一次洗涤。一般是足够的，只有50％的未结合的RNA沉淀氏s将含有&gt; 80％的原料。 <ol><li>加入等体积的异丙醇（无盐需要添加洗涤缓冲液含有1M NaCl的）。 </li><li>在20000×g离心20分钟，在4℃下弃上清。 </li><li>加入等体积的75％乙醇，离心，在20,000×g下在4℃下10分钟，弃去上清液。 </li><li>短暂离心，用200微升吸管，去除残留的乙醇。 </li><li>短暂离心，去除残留的乙醇与20微升吸管。 </li><li>不要让RNA干。重悬在100微升H 2 O的拌匀上下吹打5 - 6倍。孵育在65℃下振摇10分钟，并直接传送到冰。 </li><li>检查RNA质量通过electrophoretical分析的排除RNA降解。 </li></ol>

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Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources.</a> Biotechniques. 19, 942-945 (1995).</li><li>Friedel, C. C., D&#246;lken, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+tagging+and+purification+of+nascent+RNA:+implications+for+transcriptomics.">Metabolic tagging and purification of nascent RNA: implications for transcriptomics.</a> Mol. Biosyst. 5, 1271-1278 (2009).</li><li>Friedel, C. C., Kaufmann, S., D&#246;lken, L., Zimmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HALO+-+A+Java+framework+for+precise+transcript+half-life+determination.">HALO - A Java framework for precise transcript half-life determination.</a> Bioinformatics. , (2010).</li></ol>

作者宣称，他们有没有竞争经济利益。

新转录RNA代谢标记大幅提高电源的高通量技术，如芯片和RNA-SEQ提供更合适的模板来解决生物学问题的兴趣。本议定书进行了广泛的优化。它允许新转录RNA&gt; 1000倍浓缩，并提供高度可重复性的结果。 4SU​​标记实验的实验设计是至关重要的新转录的RNA，将描绘仅在4SU细胞的曝光时间的实时的转录活性。如果刺激后转录率的实际变化已经消退，这些都将错过新转录RNA进行分析...

基因表达谱是一个关键的工具，用来研究细胞过程和相关的复杂的相互作用网络。 mRNA丰度的研究，通常的首选方法，以获得基本的洞察潜在的分子机制。全转录组微阵列1和，最近，新一代的RNA测序（RNA-SEQ）2-4的发展助长了这种方法。虽然这些技术已经彻底改变了我们的细胞基因表达的复杂性的理解，他们面临着由于细胞总RNA模板样本， 即内在性能的主要限制。首先，短期变动的总RNA水平不相匹配的转录率的变化，但本质上是依赖于各自的转录的RNA的半衰期。虽然五倍一个短命的成绩单， 如编码转录因子的诱导，将随时检测总RNA中在一个小时之内，同样的一项长期的成绩单， 例如编码一种代谢酶的诱导，仍将几乎看不见。另外，即使是一个完整的关机的转录率的平均基因的RNA的5个小时的半衰期（&gt; 1000倍的下调）将简单地需要5个小时，它的总的RNA水平降低仅由两方面。因此，总RNA的分析有利于短命的转录调节的检测，其中有许多编码的转录因子和基因监管的功能5。此外，调节掩盖真正的动能级联和主要信号事件的不能从二级分化的。 ，反过来，可能会导致大量的偏见在下游的生物信息学分析。其次，总RNA水平的改变不能被改变RNA的合成或腐烂。后者需要测量细胞侵袭的方法， 例如阻断transcripti使用6，放线菌素D和扩展监控正在进行的RNA随着时间的推移衰减。平均mRNA的半衰期在哺乳动物细胞中的5 - 10小时5,7，大多数基因的mRNA水平的减少小于双重转录停止在几个小时后。在这些相当小的差异，导致在大多数由于基本的数学方程的指数性质的细胞基因的mRNA的半衰期非常不精确的测量。最后，当细胞总RNA的RNA-seq中透露，大约有一半是我们的基因选择性剪接事件，底层动力学以及动力机制仍然知之甚少，指导组织和上下文特定的调控RNA加工。此外，贡献差异表达基因的RNA加工，特别是对于非编码RNA，仍有待确定。总而言之，这些限制主要障碍生物信息学动力学建模相关的分子机制。 我们最近开发出一种方法，称为高分辨率基因表达谱，以克服这些问题5,7,9。它是基于使用4 thiouridine（4SU标记），一种自然发生的尿嘧啶核苷衍生物的新转录RNA代谢标记，并提供直接访问新转录转录以最小的干扰细胞生长和基因表达（ 见图1）5， 10-12。曝光的真核细胞快速吸收4SU磷酸，磷酸化，并纳入新转录RNA 4SU结果。细胞总RNA的分离，4SU标记的RNA部分是巯基的特异性生物素化的生物素和新转录的RNA之间产生一个二硫键。 “细胞总RNA&#39;然后可以进行定量分离成标记（”新转录“）和未标记的（&#39;预existin的克）RNA具有高纯度，使用链霉亲和素包被的磁珠。最后，标记的RNA回收从有孔玻璃珠，通过简单地增加裂解二硫键的还原剂（ 如二硫苏糖醇）和释放从有孔玻璃珠的新转录的RNA。 新转录的RNA描绘每一个基因的转录活性4SU曝光的时段内。 4SU ​​标记分钟的时间表，从而提供了一个快照图片真核生物基因表达和理想的模板，下游的生物信息学分析（ 如启动子分析）。在稳态条件下可以假设的情况下，新转录/总的比例，新转录/未标记的未标记/，总RNA提供非侵入性的访问精确RNA半衰期7,13。此外，重要的是要注意4SU标记只需5分钟（5分钟4SU RNA）纯化后，新转录的RNA是年龄小于15分钟和60分钟4SU-RNA。当进行超短逐步不再4SU标记的RNA-seq的结合在一个单一的实验设置，RNA加工的动力学透露核苷酸分辨率9。最后，时间过程分析与计算模型相结合的新的转录总RNA允许RNA合成的综合分析和衰减14。 总之，该方法可以直接分析RNA的合成，处理，并在真核细胞中的降解动力学。这是适用于所有主要模式生物，包括哺乳动物，昆虫（ 果蝇 ），两栖类（ 爪蟾 ）和酵母5,15,16。这是直接兼容微阵列分析5,17，9,13,14 RNA定序，适用于体内 12,15。在这里，我们详细的方法，在培养的哺乳动物细胞中的标签，隔离和净化新转录RNA。此外，电位人的问题和缺陷进行了讨论。

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>4-thiouridine</td>
 <td>Carbosynth</td>
 <td>T4509</td>
 <td>Prepare 50 mM stock in sterile H2O, store at -20 &deg;C in aliquots of 50-500 &mu;l, discard unused reagent, do not refreeze.</td>
 </tr>
 <tr>
 <td>Trizol</td>
 <td>Invitrogen</td>
 <td>15596026 (100 ml), 15596018 (200 ml)</td>
 <td>WARNING - CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol); Store at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma</td>
 <td>372978</td>
 <td>WARNING - HAZARDOUS TO HEALTH</td>
 </tr>
 <tr>
 <td>Isopropanol</td>
 <td>Sigma</td>
 <td>650447</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium citrate, nuclease-free</td>
 <td>Sigma</td>
 <td>C8532</td>
 <td>Prepare 1.6 M stock solution using nuclease-free water.</td>
 </tr>
 <tr>
 <td>5M nuclease-free NaCl</td>
 <td>Sigma</td>
 <td>71386</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>Nuclease-free H2O</td>
 <td>Sigma</td>
 <td>W4502</td>
 <td>Make 1 ml aliquots in nuclease-free tubes.</td>
 </tr>
 <tr>
 <td>RNA precipitation buffer</td>
 <td></td>
 <td></td>
 <td>1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under strictly nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma</td>
 <td>459844</td>
 <td>Use with nuclease-free water to prepare 80% ethanol, store at -20 &deg;C.</td>
 </tr>
 <tr>
 <td>1 M nuclease-free Tris Cl, pH 7.5</td>
 <td>Lonza</td>
 <td>51237</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>500 mM nuclease-free EDTA, pH 8.0</td>
 <td>Invitrogen</td>
 <td>15575-020</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>10x Biotinylation Buffer (BB)</td>
 <td></td>
 <td></td>
 <td>100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water, make aliquots of 1 ml.</td>
 </tr>
 <tr>
 <td>Dimethylformamide (DMF)</td>
 <td>Sigma</td>
 <td>D4551</td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-Link biotin-HPDP</td>
 <td>Pierce</td>
 <td>21341</td>
 <td>Prepare 1 mg/ml stock solution by dissolving 50 mg biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 &deg;C in aliquots of 1 ml.</td>
 </tr>
 <tr>
 <td>Phase Lock Gel Heavy tubes 2.0 ml</td>
 <td>Eppendorf</td>
 <td>0032 005.152</td>
 <td>Optional for the chloroform extraction step.</td>
 </tr>
 <tr>
 <td>Zeta membrane</td>
 <td>BIORAD</td>
 <td>162-0153</td>
 <td></td>
 </tr>
 <tr>
 <td>10x Dot blot binding buffer</td>
 <td></td>
 <td></td>
 <td>100 mM NaOH, 10 mM EDTA</td>
 </tr>
 <tr>
 <td>Biotin-oligo</td>
 <td></td>
 <td></td>
 <td>5'-biotin, 25 nucleotides, any sequence</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulphate</td>
 <td>Fisher</td>
 <td>BPE9738</td>
 <td>For 100 ml 20% stock solution, add 20 g SDS to 80 ml PBS pH 7-8 and adjust volume to 100 ml. Keep all high-percentage SDS solutions above 20 &deg;C. Warm the solutions slightly should SDS precipitate.</td>
 </tr>
 <tr>
 <td>EZ-Link Iodoacetyl-LC-Biotin</td>
 <td>Pierce</td>
 <td>21333</td>
 <td>Prepare 1 mg/ml stock solution by dissolving 50 mg iodoacetyl-biotin in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 &deg;C in aliquots of 1 ml. Generates irreversible, thiol-specific biotinylation.</td>
 </tr>
 <tr>
 <td>Phosphate buffer saline</td>
 <td>Gibco</td>
 <td>10010-015</td>
 <td></td>
 </tr>
 <tr>
 <td>Dot blot blocking buffer</td>
 <td></td>
 <td></td>
 <td>Mix 20 ml 20% SDS with 20 ml 1 x PBS pH 7-8 and add EDTA to the final concentration of 1 mM.</td>
 </tr>
 <tr>
 <td>Streptavidin-horseradish peroxidase </td>
 <td>Vector Laboratories</td>
 <td>SA5004</td>
 <td>Store at -20 &deg;C. Mix 10 ml 20% SDS with 10 ml 1 x PBS. Add 20 &mu;l Streptavidin-HRP before use. </td>
 </tr>
 <tr>
 <td>ECL reagent</td>
 <td>GE Healthcare</td>
 <td>RNP2109</td>
 <td>Use following the manufacturer's instructions.</td>
 </tr>
 <tr>
 <td>Super RX, X-RA Film, 18x24 cm</td>
 <td>Fujifilm</td>
 <td>47410 19236</td>
 <td></td>
 </tr>
 <tr>
 <td>&mu;Macs Streptavidin Kit</td>
 <td>Miltenyi</td>
 <td>130-074-101</td>
 <td>Store the beads at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Sigma</td>
 <td>P1379</td>
 <td></td>
 </tr>
 <tr>
 <td>Washing buffer</td>
 <td></td>
 <td></td>
 <td>100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O.</td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>Sigma</td>
 <td>43817</td>
 <td>Prepare as 100 mM DTT in nuclease-free H2O, always prepare fresh before use.</td>
 </tr>
 <tr>
 <td>RNeasy MinElute Kit</td>
 <td>Qiagen</td>
 <td>74204</td>
 <td>Store columns at 4 &deg;C, remaining components of the kit at room temperature.</td>
 </tr>
 <tr>
 <td>1.5 ml screw-top polypropylene tubes</td>
 <td>Sarstedt</td>
 <td>72.692.005</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>2.0 ml screw-top polypropylene tubes</td>
 <td>Sarstedt</td>
 <td>72.694.005</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>50 ml tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>All solutions/reagents should be stored at room temperature unless otherwise specified.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>UV/VIS spectrophotometer</td>
 <td>Thermo Scientific</td>
 <td>NanoDrop 1000</td>
 <td>Or equivalent. Use low volume (1-2 &mu;l) for measurements of low RNA concentrations to avoid excessive sample loss.</td>
 </tr>
 <tr>
 <td>Polypropylene 15 ml centrifuge tubes</td>
 <td>VWR International</td>
 <td>525-0153</td>
 <td>In contrast to standard 15 ml tubes, these tolerate up to 15,000 &times; g</td>
 </tr>
 <tr>
 <td>High-speed centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-25</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>High-speed rotor</td>
 <td>Beckman Coulter</td>
 <td>JLA-16250</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>Adaptors for 15 ml tubes</td>
 <td>Laborger&auml;te Beranek</td>
 <td>356964</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>Refrigerated table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5430 R</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Thermomixer</td>
 <td>Eppendorf</td>
 <td>Thermomixer compact</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Magnetic stand</td>
 <td>Miltenyi Biotec</td>
 <td>130-042-109</td>
 <td>One stand holds 8 &mu;Macs columns.</td>
 </tr>
 <tr>
 <td>Waterbath</td>
 <td>Grant</td>
 <td>SUB Aqua 5</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Ultra-fine scale</td>
 <td>A&amp;D</td>
 <td>GR-202</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>E-Gel iBase Power System</td>
 <td>Invitrogen</td>
 <td>G6400UK</td>
 <td>For RNA gels; or equivalent.</td>
 </tr>
 <tr>
 <td>E-Gel EX 1% agarose precast gels</td>
 <td>Invitrogen</td>
 <td>G4020-01</td>
 <td>For RNA gels; or equivalent.</td>
 </tr>
 </tbody>
</table>

metabolic labeling of newly transcribed rna for high resolution gene expression profiling of rna synthesis, processing and decay in cell culture

全转录组的芯片和新一代测序的发展已经彻底改变了我们的细胞基因表达的复杂性的理解。随着更好地理解所涉及的分子机制，底层动力学的精确测量变得越来越重要。在这里，这些有力的方法面临的主要限制，因为他们学习的模板样本， 即细胞总RNA的内在属性。在许多情况下，细胞总RNA发生变化，要么过慢或过快，代表潜在的分子事件和动力学有足够的分辨率。此外，RNA的合成，加工，和衰减的贡献改变不容易区分。 我们最近开发高分辨率基因表达谱来克服这些限制。我们的做法是根据新转录RNA代谢标记4 thiouri用餐（因此也称为4SU标记），然后通过严格的纯化的新转录的RNA，使用硫醇特异性的生物素和链霉亲和素包被的磁珠。它是适用于范围广泛的生物体包括脊椎动物， 果蝇 ，酵母或细菌。我们成功地应用于4SU标记实时动力学研究转录因子的活动，提供精确的测量RNA半衰期，到RNA加工的动力学，并取得了新的见解。最后，计算模型可以被用来产生一个综合的，全面的分析，潜在的分子机制。

1。原料，预期产量经过1小时（小时）4SU曝光新转录RNA约占1 - 4％的细胞总RNA。这将是较低的，因为他们生长被捕细胞不再合成RNA占细胞生长/复制。贴标签时1小时，我们建议60 - 80微克总RNA开始检测。小于30微克总RNA的小RNA颗粒生物素化步骤，是很难看后开始，因此可以很容易丢失。输入RNA水平可能会增加多达150微克的持续时间很短标签（ 如 5 - 10分钟）。当RNA标记的?...

Watch this Scientific Journal Video about Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture at JoVE.com

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

细胞总RNA提供了一种用于研究的短期变化，以及在RNA合成和衰变动力学的RNA加工模板差。这里，我们描述遵循与4 - thiouridine的新转录的RNA的代谢标记通过硫醇特异性的生物素化和纯化的新转录的RNA，来克服这些限制。

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

metabolic-labeling-newly-transcribed-rna-for-high-resolution-gene

Research

JoVE Journal

Biology

27.0K 次观看.  Max von Pettenkofer Institute. 细胞总RNA提供了一种用于研究的短期变化，以及在RNA合成和衰变动力学的RNA加工模板差。这里，我们描述遵循与4 - thiouridine的新转录的RNA的代谢标记通过硫醇特异性的生物素化和纯化的新转录的RNA，来克服这些限制。

视频: Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

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

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

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

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

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

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...