国防部（创意奖W81XWH-07-0406） - 乌鲁斯阿塔索伊 NIH RO1 A1080870 - 乌鲁斯阿塔索伊 NIH R21 A1079341 - 乌鲁斯阿塔索伊密苏里州的机构资金 - 乌鲁斯阿塔索伊

实验准备在开始实验之前，这是关键的是具有所有试剂，容器和用具，无RNase。治疗玻璃器皿与RNase抑制剂（RNaseZAP，Ambion公司），然后用DEPC处理过的水冲洗。确保所有的试剂被确认为无RNase。 1。准备mRNP裂解液<ol><li>种植和收获成倍增长的组织细胞产生的总蛋白2-5毫克之间，每个RIP。 <ol class="lalpha"><li>两个P150培养皿通常就足够了。 </li><li>对于每个被调查的RBP，总细胞数和蛋白质的量必须进行优化，以最大限度地提高相应的靶mRNA和RBP的相互作用。 </li><li> RIP定量RT-PCR分析，可能需要较少的细胞裂解液（约400μg总蛋白），由于HuR的TIAR，AUF1，尤其是对高丰度的限制性商业惯例，其扩增和检测方法。 </li></ol> &lt;/ P&gt; <li>颗粒细胞通过离心200×g离心8-10分钟，在4°C，然后用冰冷的PBS洗三次。 </li><li>最后一次洗涤后，轻轻地滑动管的底部和重悬细胞沉淀，在制备的多核糖体RNase及蛋白酶抑制剂的裂解缓冲液（PLB）与等体积的。 <ol class="lalpha"><li>建议测量的准确体积的细胞沉淀。 </li></ol></li><li>轻轻地移液管混合（涡），掰开细胞团块，和裂解液在冰上孵育5分钟。 </li><li>离心机在13,000 xg离心20分钟，在4℃至清除杂物的溶胞产物。 </li><li>立即转移到预冷的微量离心管中清零上清液。 </li><li>查看在冰上并储存在-80℃下<ol class="lalpha"><li>立即冻结完成适当的裂解细胞，并防止不必要的绑定。继续与RIP紧随其后的样品融化和冰样品，以避免RNA的降解。/ P&gt; <li>此溶胞产物可以存储最多六个月在-80℃下应避免反复冻融循环，因为这可以导致蛋白质和/或mRNA的降解。 </li></ol></li><li>在使用标准布拉得福德蛋白质分析的溶胞产物，测定蛋白质浓度。 </li></ol> 2。外壳蛋白A琼脂糖珠与抗体和冲洗<ol><li>预膨胀蛋白A琼脂糖凝胶（PAS）珠过夜，用5％BSA在NT2缓冲（3-4卷）。存放于4℃ <ol class="lalpha"><li>长期贮存几个月在4°C是可能的时，补充有0.1％的叠氮化钠。 </li><li>目标RBP抗体同种型的基础上，应选择蛋白质Sepharose磁珠。蛋白A，G和A / G都有特定的抗体亚型目标和不同的目标亲和力。蛋白A琼脂糖珠的基础上同型HuR的蛋白抗体的特异性和亲和力。 </li></ol></li><li>前使用，去除多余的的NT2缓冲，所以最终珠缓冲区比为1:1。 </li><li>用1.5ml无RNA酶的离心管中，取出100微升的PAS浆，加入30微克的抗体反应（ 即 ，具体RBP及同型对照）每个IP。 </li><li>加入100-200微升NT2缓冲抗体珠组合。 <ol class="lalpha"><li>混合物可被存储为几个星期，在4℃时，补充有0.1％的叠氮化钠。 </li><li>同种型匹配的抗体或来自同一物种的整个正常血清应并行地使用在背景RNA作为抗体，该抗体控制。 </li></ol></li><li>珠混合添加相应的抗体，孵育过夜，比上年末翻滚在4°C。 <ol class="lalpha"><li>优化被调查的特定蛋白的抗体效价（1，5，10或30μg的抗体通常是足够的）。 </li></ol></li><li>准备使用前立即用1ml的洗涤的抗体包被的珠子冰冷却NT2缓冲器5倍。 <ol class="lalpha"><li>通过离心1-2分钟，在4℃下以13,000 xg离心珠混合洗净</li><li>小心地取下的最高金额supernantant手动移液器或吸气，但要小心，以避免干扰颗粒。 </li><li>洗衣机有助于除去未结合的抗体以及抗体混合物的RNA酶污染物。 </li></ol></li><li>一旦已经完成最后一次洗涤后，重悬在700微升冰冷的NT2缓冲区后跟治疗与各种RNase抑制剂的保护的靶mRNA，包括10微升的RNaseA暂在40单位/微升，10微升的100mM珠DTT和15μlEDTA（15毫米）。把音量到1000微升NT2缓冲。 <ol class="lalpha"><li>氧钒核糖核苷复合物由于不使用EDTA的抑制作用。 </li></ol></li></ol> 3。免疫沉淀和RNA沉淀<ol><li>可选的预清除步骤:<ol class="lalpha"><li>以减少背景，珠子和对照抗体，也可以使用预先明确的溶胞产物。这可能会降低信号在输出中。 </li><li>这一步可能需要减少的背景时，IP其次是芯片。它通常是没有必要的IP定量RT-PCR。 </li><li>预清除15微克的同型对照为30分钟/ 4°C比上年末翻滚。 </li><li>加入50μlpre​​swollen PAS珠涂有抗体的步骤2.1。 </li><li>孵育30分钟，比上年末/ 4°C旋转结束</li><li>在4℃下以10,000 xg离心保存上清IP。 </li></ol></li><li>加入100μl的隔离澄清的裂解液（约2-5毫克）制备的抗体组合。 <ol class="lalpha"><li>稀释裂解，将有助于减少背景和非特异性结合。 </li><li>裂解液输入量可能会有所不同，具体取决于检测方法和丰富的RBPØŕ效率RBP抗体所指出的步骤1.1.c. </li></ol></li><li> （可选）立即混合管轻轻轻弹数次，然后短暂离心，以10,000 xg于4℃下沉淀珠，并立即取出100μl的上清液作为输入总mRNA的表示qPCR分析使用标准的RNA分离技术。 <ol class="lalpha"><li>这一步是确认输入裂解RNA是最佳的IP，应该只执行一个检查步骤或差的RNA结果RIP后的故障排除步骤。 </li></ol></li><li>换行管封口膜，以确保密封和在4°C孵育2〜4小时，比上年末翻滚。 <ol class="lalpha"><li>优化的基础上孵化的时间应该在目标丰度，应尽量避免复杂的重排或退化。对于某些限制性商业惯例较短孵育可能会更理想。 </li></ol></li><li>颗粒珠在5000 XGF或5分钟，在4°C，保存上清潜力分析Western blot检测。将等分试样储存于-80℃。 <ol class="lalpha"><li>残余靶蛋白具有高量的等分的上清液可以指示故障的蛋白质沉淀琼脂糖珠。 </li></ol></li><li>如前所述，洗珠5次用1ml冰冷NT2缓冲液和离心（5,000×g离心5分钟，在4℃），然后去除上清液与手移液器或一个吸气。 <ol class="lalpha"><li>更严格的洗涤方法，可以使用以降低背景补充NT2脱氧胆酸钠缓冲液，尿素或SDS。 </li><li>样本保持尽可能多的冰和快速工作，以减少降解靶mRNA。 </li></ol></li><li>可选:一个小试样（10％），珠混合并行运行更多的样品检查IP效率的目标蛋白，用Western blot分析。</li></ol> 4。 DNA酶和蛋白酶K的治疗<ol><li>洗涤后，用100μl的NT2缓冲液重悬珠补充用5μl的无RNA酶的DNA酶1（2 U /微升）。 </li><li>请在37℃下5-10分钟。加入1毫升的NT2缓冲区，并在5,000 xg离心1分钟，在室温下离心。 <ol class="lalpha"><li>可以使用一个小的（〜10％）的等分试样的珠子来检查IP通过SDS-PAGE分析的效率。 </li></ol></li><li>首席助理局长沉淀重悬在100μlNT2缓冲液，5微升的蛋白酶K（10毫克/毫升），和1μl10％SDS。 </li><li>孵育30分钟，再悬浮的胎圈混合物在55℃下在水浴中，在每10分钟轻轻轻弹。 <ol class="lalpha"><li>蛋白酶K，将有助于在释放的RNP组件。 </li></ol></li><li>佩莱珠在5,000 xg离心5分钟，在室温（RT），并收集上清液（约100微升）。 </li><li>加入200μlNT2缓冲珠，离心华夫格5000×g离心2分钟，在室温下，收集上清液（约200μL），并丢弃珠。 </li><li>合并上清液（加入100μl和200μl），并添加300微升的酸苯酚-CHCl 3下层。 </li><li>涡，1分钟室温（或37℃下在摇动器）和在RT下以16,000 xg离心1分钟的离心分离机。 </li><li>收集250微升的上层（不破坏接口），加入25μL醋酸钠在pH值5.2，625μL100％的乙醇和5μlglycoblue，拌匀。 </li><li>贮存于-20℃下过夜<ol class="lalpha"><li>的RNA，以确保适当的恢复，此外glycoblue增加的可见性通过作为载体分子的RNA沉淀。 </li></ol></li><li>翌日，混合管由反演的3-5倍，自旋以12,000 xg于4℃下30分钟，并弃去上清液。 </li><li>加入1毫升70％ETOH蓝色的颗粒和通过反转或涡旋混合。 </li><li>离心机在4℃下12,000 xg离心2分钟。 </li><li> ðiscard上清液和12,000 xg下离心1分钟，在4℃。 </li><li>除去任何残余的70％ETOH于RT的移液管和空气干燥沉淀5分钟。 </li><li>重悬在20〜40μl的RNA酶/无DNA酶水或其他适当的音量。 <ol class="lalpha"><li>现在已准备好进一步向下游应用，如实时定量RT-PCR或芯片的样品。 </li><li>的NanoDrop分光光度法可用于评估样品的浓度，然而，贵金属的样品中，它可能是有利的避免nanodropping样品，因为它可以是浪费和相对不准确的。我们建议正常化样本，以看家基因转录宝石或低收益率样本的纯度和IP效率评估。 </li></ol></li></ol> 5。代表性的成果如果程序进行了优化，并进行正确的免疫沉淀产生显着富集的mRNA靶标。通常情况下，根据RBP和它的靶mRNA（S），我们可以看到进行评估时，通过实时定量RT-PCR富集约10 - 50倍。许多限制性商业惯例的目标可以发现集体使用基因芯片分析。然而，这种方法是用实时定量RT-PCR方法相比更敏感的降解。根据上RBP，目标的数量和反应效率，微阵列可以揭示数百个新的目标，或者它可以只发现了很少，如果有的。例如，一个更好的特点RNA结合蛋白HuR的，转录后调控许多重要的生理基因1，2的表达和翻译。通过RIP芯片在乳腺癌细胞株中的HuR的ribonucleo复杂的分离，例如，显示的几个重要的已知HuR的目标，包括使用定量RT-PCR方法，如在图1中所示的β-肌动蛋白的富集。在这两种肿瘤细胞株的β-肌动蛋白富集的12 - 至15 - 倍。通常情况下，正确执行时，我们看到了显着的ENRichment各种细胞系中的β-肌动蛋白。然而，如果RIP没有发现任何显着的β-肌动蛋白的富集这表明与RIP的一个问题，可能需要重复该过程。此外，从这些细胞系的免疫沉淀的样品的微阵列分析的子集显示不同的表达HuR的目标在不同的雌激素受体 ​​（ER）阳性的MCF-7癌细胞与ER阴性MB-231乳腺癌细胞株作为相比，表明在图2 1。这些目标分为几类:已知和未知的HuR的目标，无论是与癌症有关或无关的。例如，CALM2和CD9都是以前没有HuR的目标确定为癌基因。使用微阵列和确认用定量RT-PCR，CALM2和CD9被发现是5 - 至180倍富集HuR的粒料中的指示突出HuR的蛋白质和这些目标基因之间的相互作用。 <i毫克SRC ="所有/ files/ftp_upload/3851/3851fig1.jpg"ALT ="图1"/&gt; 图1。免疫沉淀和RIP MB-231（ER）和MCF-7（ER +）乳腺癌细胞 MB-231，MCF-7细胞裂解液进行免疫沉淀。使用抗HuR的单克隆抗体（3A2）和IgG1同型对照。 A. IP西户珥透露，预计大小的带检测3A2。在右边的面板上显示金额，户珥的输入裂解液使用这两个细胞系，与β-微管蛋白作为加载控制。 B.定量RT-PCR验证表明，3A2 IP地址从MB-231和MCF-7，15和11倍的富集β-肌动蛋白，已知的HuR的目标。 ΔΔCT值进行标准化，以GAPDH。进行了实验，一式两份，每份（n = 2时）。 <img src="/files/ftp_upload/3851/3851fig2.jpg" alt="图2" /> 图2。 HuR的RIP-CHIP确定不同的基因图谱，在ER +和ER-的乳腺癌细胞。HuR的免疫沉淀进行从MB-231或MCF-7细胞的裂解物使用的HuR抗体和IgG1同种型对照的Illumina公司Sentrix阵列（47,000个基因）杂交。控制信号中减去。结果代表来自12个不同的阵列的累计数据。实验进行了每种细胞系相匹配的控制组（n = 3），一式三份。秤是LOG2。

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+Regulatory+Mapping+Indicates+that+the+RNA-Binding+Protein+HuR+Couples+Pre-mRNA+Processing+and+mRNA+Stability.">Integrative Regulatory Mapping Indicates that the RNA-Binding Protein HuR Couples Pre-mRNA Processing and mRNA Stability.</a> Molecular Cell. 43, 1-13 (2011).</li></ol>

作者宣称，他们有没有利益冲突。

由于这个实验的性质，优化和经验的唯一可靠的方法，成功地获得预期的结果。在此过程中的许多步骤，温度和所用试剂和产品的高效的处理是极为重要的。适当的规划和执行的技术将有助于确保该实验是在一个合理的时间在推荐的最佳温度。 RNA分离实验的一个主要问题是由核糖核酸酶的敏感性RNA的降解。所有的试剂需要无RNA酶的无RNase的容器中并贮存或使用。这是一个关键的步骤，以确保你的基因样本的完整性。甚至当实验正确执行，然而，期望的结果可能无法实现由于RBP和其靶mRNA之间的相互作用的性质。 一个潜在的问题是有低，甚至无信号提取的RNA RIP-芯片。虽然有可能是从总RNA中的信号，这可能是由珠子被拉下的结合蛋白不足的结果。第一个故障排除步骤是确认的细胞裂解液正在使用的具体RBP有足够的表达。经确认后，蛋白质可被分离后最终NT2洗涤并重新悬浮在Laemmli缓冲或另一种合适的变性缓冲液，并加热，在95℃的5分钟。 Western blot分析可以用在这些样品中的协调与输入裂解液作为阴性对照，以确保有足够的下拉相关蛋白。 此外，因为裂解细胞需要访问这些组件，通常分离的蛋白质和mRNA的异常和不必要的相互作用的潜在可能被引入。这些相互作​​用可能与"吸收"靶mRNA结合蛋白通过非特异性相互作用。此外，蛋白质在这些不同的合作条件可以折叠的多种变体，其结合基序可能变得不可访问他们的目标基因，防止它们之间的相互作用。这些都加强了工作效率，以及利用最佳温度限制这些有害的相互作用的重要性。此外，为每个特定的目标蛋白的洗涤条件的优化将是至关重要的最大化的相互作用的纯度。可能需要更严格的洗涤条件。例如，洗涤缓冲液可以被补充与SDS或一个适当的量的尿素，以减少非特异性的相互作用和背景中的信号输出。这将是完全依赖于实验者的目标的RBP以及作为其独特的生理条件下与靶mRNA中。在某些情况下，将不适合于某些mRNA的分析工具，应当指出，在制备的样品。 最后，虽然在富集的RNA RIP是成功-RBP的互动与RIP（CHIP）的方法，一个众所周知的问题是无法确定具体的RBP结合域的瞬时表达目标。几种交联技术可用于随后RIP隔离独特的序列目标，但是，使用短波UV趋于导致核酸损伤。一种新方法被称为的PAR-CLIP，或光活化的核糖核苷的交联剂和immuoprecipitation，采用长波UV纳入thiouridine成新生RNA允许从稳定和瞬时RNA的相互作用的独特的结合位点的识别。 总体而言，RIP-芯片已经建立了一个很好的工具，用来分离和研究组，以及许多其他的研究小组之间的相互作用RNA结合蛋白和mRNA的目标。虽然敏感的性质和实践，正确执行此过程会产生这些RNP复合物隔离，直到最近，已经INACC的发现和分析essible为。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 1 M二硫苏糖醇</td><td>费舍尔</td><td> BP172-5 </td><td> DTT </td></tr><tr><td> DNA酶1 </td><td> Ambion公司</td><td> 2235 </td><td>无RNase </td></tr><tr><td>乙二胺Tetraccetic酸</td><td>费舍尔</td><td> BP118-500 </td><td> EDTA </td></tr><tr><td>糖原</td><td> Ambion公司</td><td> 9516 </td><td></td></tr><tr><td> 1 HEPES </td><td>西格玛</td><td> H3375-100G </td><td> pH值7.0 </td></tr><tr><td>的Igepal NONIDET P-40 </td><td> USB </td><td> 78641 </td><td> NP40 </td></tr><tr><td> 1 M氯化钾</td><td>费舍尔</td><td> BP366-500 </td><td></td></tr><tr><td> 1 M的MgCl 2 </td><td>费舍尔</td><td>乙P214-500 </td><td></td></tr><tr><td> NT2缓冲区</td><td>见下面</td><td></td><td></td></tr><tr><td>多聚核糖体裂解缓冲液</td><td>见下面</td><td></td><td></td></tr><tr><td>蛋白酶抑制剂鸡尾酒粒的</td><td>罗氏公司</td><td> 11873580001 </td><td></td></tr><tr><td>蛋白A Sepharose磁珠</td><td>西格玛</td><td> P3391 </td><td></td></tr><tr><td>蛋白酶K </td><td>费舍尔</td><td> BP1700-100 </td><td></td></tr><tr><td>核糖核酸酶出核糖核酸酶抑制剂</td><td> Invitrogen公司</td><td> 10777-019 </td><td> 40 U /μL </td></tr><tr><td> 1M NaCl的</td><td>费舍尔</td><td> BP358-212 </td><td></td></tr><tr><td>十二烷基硫酸钠</td><td>费舍尔</td><td> BP166-500 </td><td> SDS </td></tr><tr><td> 1 M的Tris-HCl </td><td>费舍尔</td><td&gt; BP153-500 </td><td> pH值7.4 </td></tr><tr><td> Trizol法</td><td> Invitrogen公司</td><td> 15596-026 </td><td></td></tr><tr><td>钒核糖核苷复合物</td><td>新英格兰实验室</td><td> S1402S </td><td> VRC </td></tr></tbody></table> <table border="1"><tbody><tr><td> 试剂全面评估 </td></tr><tr><td>核糖核酸酶/无DNase的，DEPC处理过的玻璃器皿中制备试剂</td></tr><tr><td> 多聚核糖体裂解缓冲液 </td></tr><tr><td> 100毫米氯化钾</td></tr><tr><td> 5毫摩尔MgCl 2 </td></tr><tr><td>的10mM HEPES（pH7.0）中</td></tr><tr><td> 0.5％NP40 </td></tr><tr><td> 1毫米DTT </td></tr><tr><td> 100单位/毫升核糖核酸输出</td></tr><tr><td> 400μMVRC </td></tr><tr><td>蛋白酶抑制剂鸡尾酒平板的</td></tr><tr><td> 5毫升的多核糖体的裂解缓冲液 </td></tr><tr><td>添加50μl的1 M HEPES（pH值7.0） </td></tr><tr><td> 500μl的1 M KCL </td></tr><tr><td> 25微升的1 M的MgCl 2 </td></tr><tr><td> 25μl的NP40 </td></tr><tr><td> 4.7毫升无RNA酶的DNA酶H 2 O </td></tr><tr><td> 50微升的1 M DTT </td></tr><tr><td> 100 U / ml的RNA酶12.5μl的出</td></tr><tr><td> 200μl蛋白酶抑制剂混合物（溶于根据制造商） </td></tr><tr><td> 10微升的200mM VRC（在使用时） </td></tr><tr><td> NT2缓冲区 </td></tr><tr><td>的50mM的Tris-HCl（pH7.4）中</td></tr><tr><td> 150mM NaCl的</td></tr><tr><td> 1毫摩尔MgCl 2 </td></tr><tr><td> 0.05％NP40 </td></tr><tr><td> 1 L的NT2缓冲器 </td></tr><tr><td>的50毫升Tris（pH7.4）中</td></tr><tr><td> 30毫升5 M氯化钠</td></tr><tr><td> 1毫升1 M氯化镁<suB&gt; 2 </td></tr><tr><td> 500μLNP40 </td></tr><tr><td> 820毫升RNA酶，无DNA酶H 2 O </td></tr></tbody></table>

method for the isolation and identification of mrnas, micrornas and protein components of ribonucleoprotein complexes from cell extracts using rip-chip

作为发展的高通量测序和高效的微阵列分析的结果，全球基因表达分析已经成为一个简单的数据收集和随时可用的形式。在许多研究，然而，疾病模型，靶基因mRNA的稳态水平不总是直接与稳态蛋白水平。基因转录后调控是一个可能的解释两者之间的分歧。结合的RNA结合蛋白（RBP）的带动下，形成核糖核蛋白（RNP）复合物与靶mRNA的转录后调控影响mRNA的定位，稳定性和翻译的。确定这些未知从头目标mRNA的细胞提取物中的RNP复合物的RBP和其造成的影响对蛋白质的输出理解的机制和功能的关键。该协议概述的方法被称为RNP免疫沉淀芯片（RIP-Chip）的，允许s的识别pecific的mRNA的核糖核蛋白复合物，在试验条件下，随着选项，以进一步优化的个别研究者的实验。有了这个重要的实验工具，研究人员能够探索与转录后基因调控，以及其他核蛋白的相互作用的复杂机制。

Watch this Scientific Journal Video about Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip at JoV

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

一步一步协议通过RIP-芯片的分离和鉴定RNA相关的复合物。

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

method-for-isolation-identification-mrnas-micrornas-protein

Research

JoVE Journal

Biology

27.5K 次观看.  University of Missouri. 一步一步协议通过RIP-芯片的分离和鉴定RNA相关的复合物。

视频: Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

遗传学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

生物学

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.