The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

注:关键是要在整个过程中保持无RNA酶的条件。 1.腺苷酸化的3&#39;连接器<ol><li>稀释设计用于3&#39;连接反应至100μM在不含核酸酶的H 2 O的DNA寡核苷酸 注:该寡核苷酸应具有一个5&#39;磷酸基团，将在5随机化的核苷酸&#39;末端和3&#39;dideoxycytosine。 5&#39;磷酸基团是第M个酶的高效5的规定&#39;腺苷酸化10。磷酸盐可以通过预处理寡核苷酸的添加与多核苷酸激酶，或排序，直接修改。随机核苷酸在5&#39;端是用于最小化序列偏好其中的RNA连接酶表现出对于其他6某些端到端连接的反应很重要。在3的寡核苷酸吨端&#39;dideoxycytosine在3放置一个阻挡元件"ø抑制的接头-连接体多联体在随后的连接步骤的形成和还用于阻止连接反应11 3&#39;形成于3的端部与miRNA -接头分子的端部"。选择用于无偏的miR-SEQ方法的寡核苷酸接头序列如下:5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3&#39;。 </li><li>组装下面腺苷酸化反应:200pmol的3&#39;接头寡核苷酸，4微升10×5&#39;腺苷酸化的DNA缓冲液，4μl1mM的ATP，4μl的第M个 RNA连接酶，核酸酶游离H 2 O至40微升的最终体积。 </li><li>孵育在65℃下反应1小时。终止反应，加热至85℃5分钟。 </li><li>通过加入2.5倍体积的100％乙醇和1/3体积的10M乙酸铵沉淀腺苷酸化的连接体，以除去残留的ATP，并防止在将来的反应未预料到的连接反应。 <ol><li>简单地说，混合人cohol，盐，并在4℃下进行20分钟的寡核苷酸溶液至均匀，以全速在微量离心纺丝（〜15000×g离心）。洗70％的乙醇，空气干燥，悬浮颗粒在核酸游离H 2 O的适当体积，以产生50-100微米寡腺苷酸化的。 </li></ol></li><li>通过运行在18％聚丙烯酰胺凝胶确认腺苷酸化。 <ol><li>通过混合制备凝胶下面一起6.3克尿素，6.75毫升40％的丙烯酰胺，和1.5毫升10×TBE。加入H 2 O操作15毫升。 </li><li>混合直到尿素完全溶解，并添加150μl的10％（重量/体积）过硫酸铵（APS）和15微升tetramethlyethylenediamine（TEMED）。 </li><li>一旦凝胶已设定，预运行30分钟，在凝胶宽度的24伏/厘米用1×TBE缓冲液在室温下。一旦30分钟可达冲洗井用电泳缓冲液。 </li><li>拌5皮摩尔腺苷酸化寡核苷酸与和的2倍变性的RNA上样缓冲液等体积​​（95％（体积/体积）甲酰胺，0.02％（重量/体积）SDS，0.02％溴酚蓝（重量/体积），0.01％（重量/体积）二甲苯蓝，1.0mM的EDTA）中，热简要至75℃，并搭扣在冰上冷却。分配整个样品进入凝胶的孔中。还包括一个相同的量的非腺苷化控制的，以及低分子量大小标准。 </li><li>运行该凝胶在24伏/厘米，直到溴酚蓝的方式向凝胶的底部¾。以在1×TBE缓冲液1×SYBR -金溶液拆卸装置，后染色凝胶和可视化的紫外透射仪箱（ 图2）。 注:凝胶纯化寡腺苷酸化在类似于上面描述的，如果"没有输入的RNA"控件都导致高的量的PCR产物的方式相比，与输入的RNA样品。 </li></ol></li></ol> 2.连接器结扎到的miRNA的3&#39;末端<ol><li> 3&#39;结扎<ol><li>装配在冰上下列组分按所列顺序:1.0微升50％（重量/体积）的PEG 8000，0.5＆＃956;微升10x RNA连接酶缓冲液（500毫摩尔的Tris-HCl pH为7.5，100毫摩尔的MgCl 2，10毫摩尔DTT），1.0微升腺苷酸化的3&#39;连接器（100μM），0.5μl的RNase抑制剂，0.5-8.0微克总RNA， 0.5微升T4 RNA连接酶2，无核酸酶的H 2 O操作的5微升的最终体积。 </li><li>混合通过移液反应的溶液太粘稠，旋涡，由于高浓度的PEG 8000。一旦彻底混合，将用加热盖在热循环仪中进行反应。设置块℃至16℃，并保温4小时。 注:该反应可以按比例增加至20微升的最终体积，而不在连接效率的任何损失。反应4小时，在环境温度下过夜或在4℃下，以产生基本上相同的连接效率。 </li></ol></li><li>凝胶净化<ol><li>继3&#39;结扎，拌上2×RNA上样缓冲液等体积​​反应。热至70℃5分钟，将其卡在冰上冷却。 </li&gt; <li>运行用于PAGE纯化15％聚丙烯酰胺凝胶中的样品。 <ol><li>制备含有7M尿素的15％聚丙烯酰胺凝胶，在类似于步骤1.5先前描述的方式。 </li><li>运行前的凝胶在凝胶宽度的24伏/厘米至少30分钟。关闭电源，冲洗水井，负载样品和凝胶运行在相同的电压预运行。 </li><li>装载样品和运行页面，直至溴酚蓝刚退出凝胶。所需产物将迁移靠近二甲苯蓝。 </li></ol></li></ol></li><li>染色，可视化，并切除<ol><li>在干净的托盘1X电泳缓冲液（TBE），1X SYBR金，和摇滚15分钟拆卸胶设备和场所凝胶。 </li><li>从染色皿中取出凝胶和可视化的紫外透射盒子盖上干净的保鲜膜。连接产物应该是大约45个核苷酸长度。 </li><li>切下含有连接产物的区域用一块干净的razo- [R刀片和地点在0.5ml微离心管中。 </li></ol></li><li>产品分离和降水<ol><li> 0.5 ml离心管刺穿底部与地下30针，然后将穿孔的管中的第二清洁，低滞留，1.5 ml离心管。离心5分钟，在〜15,000×克。 </li><li>目视确认该整个凝胶切片已经通过在所述内管的孔移动。如果有必要，用干净的枪头一些凝胶碎片距离拉得更近了洞。 </li><li>弃去空0.5毫升管中，加入400μl的高盐柱缓冲液（HSCB 400 mM氯化钠，的25mM的Tris-HCl pH为7.5，0.1％SDS）中的丙烯酰胺的浆液。岩管（多个）在4℃下过夜。 </li><li>使用宽孔移液管尖端传送浆料到0.22微米醋酸纤维素离心柱上。离心3分钟，在室温下以〜15000×g离心，收集所有的液体远离丙烯酰胺凝胶基质。 </li><li>液体转移到一个干净的，低保留，1.5毫升试管中含有荷兰国际集团1微升20mg / ml的糖原。添加异丙醇等体积的1/10体积乙酸钠。通过倒转试管几次混合溶液和沉淀在-80℃下冷冻20分钟。 </li><li>离心机以全速在4℃下进行20分钟以沉淀核酸。除去上清，洗涤在70％乙醇中。空气干燥沉淀物，在室温下10分钟，并直接进行5&#39;连接步骤。 </li></ol></li></ol> 3.连接器连接以5&#39;端的miRNA的3&#39;混合连接器<ol><li>以沉淀核酸，添加以下组分:2微升PEG 8000（50％重量/体积），0.5微升10×RNA连接酶缓冲液，0.5微升ATP（10毫摩尔），0.5微升NN-RNA的寡核苷酸（100μM）和H 2 O 4微升的最终体积。 注:"NN-RNA的寡核苷酸"的组合物是如下5&#39;-倒置脱氧TCUACAGUCCGACGAUCNN3&#39;和由生产经HPLC进行纯化。 </li><li>混此示例吹打上下反复。这样做一段时间（最长5分钟），以确保颗粒被完全重新溶解。如果再溶解是有问题的，加热样品简短（3-5分钟）至37℃，并继续移液混合。 </li><li>一旦粒料是早在溶液中，并将内容物转移到含有1微升1干净的PCR管中:RNA酶抑制剂和T4 RNA连接酶1（5个单位）的1:1混合物。通过上下吹打再次混合。 </li><li>孵育在热循环仪中，反应在37℃下搅拌4小时。立即进行cDNA合成。 </li></ol> 4.反转录/ cDNA合成<ol><li>将以下组件添加到5&#39;连接反应:2微升5×RT缓冲液，0.75微升100mM的DTT，1微升Illumina公司的RT引物5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39;（1μM），和0.5微升的dNTPs（10毫摩尔）。 </li><li>通过移液和热拌至65℃5分钟。然后慢慢冷却在板凳吨同前。 </li><li>一旦反应冷却至室温，放置​​管在冰上并添加每个逆转录酶和RNA酶抑制剂0.5微升。通过移液并将其放置在用于制造商的指示所指示的延伸步骤的热循环仪中混合。 </li><li>一旦完成添加5微升不含核酸酶的H 2澳带来的cDNA文库的最终体积达到15微升。 </li></ol> 5.图书馆准备<ol><li>为索引的PCR，装配在冰上下列反应:6微升5倍的Phusion缓冲液，1微升的dNTPs（10毫摩尔），0.9微升的DMSO，0.75μl的5&#39;引物（25μM），0.75μl的3&#39;引物（25μM）， 3微升cDNA文库（取决于输入的量可变浓度），加入0.5μlPhusion聚合酶（1单位），无核酸酶的H 2 O操作的30微升的最终体积。选择的引物序列，以确保与模板和测序平台的兼容性的选择。 <ol><li>运行，使用以下设置将PCR反应:在98℃预变性3分钟，在第4个周期，变性98℃15秒，退火50℃15秒，72℃延伸15秒。 注:根据所输入的材料的量，改变总的PCR循环数。典型地，对于1-2微克总RNA的一个输入端，13个总PCR循环足以产生所述的miR-SEQ库。 </li><li>进行随后的周期中， 例如，循环5-12中，在退火和延伸循环相结合，并在70℃下30秒和变性的两步方式是再次98℃持续15秒。 注:反应体积为30微升，以允许暂停PCR程序，并删除一个10微升等分试样在预先确定的周期数来监视外观所需产物。通常情况下，循环10，13，和16被选择，用于从总RNA样品的测试。 </li></ol></li><li>准备通过混合2毫升40％的丙烯酰胺，加入1ml的10×TBE，和H 2 O至10ml 8％非变性聚丙烯酰胺凝胶。然后加入100微升10％APS和10微升TEMED的。倒入凝胶，让集。 <ol><li>运行前的凝胶在凝胶宽度的12伏/厘米30分钟。同花顺井。加入1微升10×加样缓冲液（15％聚蔗糖-400，66毫摩尔EDTA，20毫摩尔的Tris-HCl pH 8.0的，0.1％（重量/体积）SDS，0.01％（重量/体积）溴酚兰）中，添加10微升的样品尺寸的各孔5毫米×1毫米×15毫米。典型地，每个孔可以加载多达30微升的样品。运行示例凝胶在凝胶宽度在1×TBE缓冲液中10伏/厘米，直至溴酚蓝染料刚刚跑出了凝胶的底部。 </li><li>染色，如在步骤2.3中所述可视化凝胶。 </li><li>消费税的146碱基对带和洗脱过夜0.25毫升HSCB在类似于在2.4节中描述的一种时尚。在异丙醇中的等体积的1/10体积乙酸钠使DNA沉淀。洗涤沉淀在70％Ethanol并悬浮在TE缓冲液。 </li><li>使用的PicoGreen根据制造商的建议进行量化的库。 </li></ol></li></ol>

<ol>
	<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-ligase-dependent+biases+in+miRNA+representation+in+deep-sequenced+small+RNA+cDNA+libraries.">RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.</a> RNA. 17 (9), 1697-1712 (2011).</li><li>Zhang, Z., Lee, J. E., Riemondy, K., Anderson, E. M., Yi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+RNA+cloning+enables+accurate+quantification+of+miRNA+expression+by+deep+sequencing.">High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing.</a> Genome Biology. 14 (10), 109 (2013).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ENCODE+(ENCyclopedia+Of+DNA+Elements)+Project.">The ENCODE (ENCyclopedia Of DNA Elements) Project.</a> Science. 306 (5696), 636-640 (2004).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Linsen, S. E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+and+possibilities+of+small+RNA+digital+gene+expression+profiling.">Limitations and possibilities of small RNA digital gene expression profiling.</a> Nature Methods. 6 (7), 474-476 (2009).</li><li>Jayaprakash, A. D., Jabado, O., Brown, B. D., Sachidanandam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+remediation+of+biases+in+the+activity+of+RNA+ligases+in+small-RNA+deep+sequencing.">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.</a> Nucleic Acids Research. 39 (21), 141 (2011).</li><li>McLaughlin, L. W., Romaniuk, E., Romaniuk, P. J., Neilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Acceptor+Oligoribonucleotide+Sequence+on+the+T4+RNA+Ligase+Reaction.">The Effect of Acceptor Oligoribonucleotide Sequence on the T4 RNA Ligase Reaction.</a> European Journal of Biochemistry. 125 (3), 639-643 (1982).</li><li>Zhuang, F., Fuchs, R. T., Sun, Z., Zheng, Y., Robb, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bias+in+T4+RNA+ligase-mediated+3&#8242;-adapter+ligation.">Structural bias in T4 RNA ligase-mediated 3&#8242;-adapter ligation.</a> Nucleic Acids Research. 40 (7), 54 (2012).</li><li>Harrison, B., Zimmerman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-stimulated+ligation:+enhanced+ligation+of+oligo-+and+polynucleotides+by+T4+RNA+ligase+in+polymer+solutions.">Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.</a> Nucleic Acids Research. 12 (21), 8235-8251 (1984).</li><li>Torchia, C., Takagi, Y., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+RNA+ligase+is+a+homodimeric+protein+that+catalyzes+intramolecular+ligation+of+single-stranded+RNA+and+DNA.">Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.</a> Nucleic Acids Research. 36 (19), 6218-6227 (2008).</li><li>Lau, N. C., Lim, L. P., Weinstein, E. G., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Abundant+Class+of+Tiny+RNAs+with+Probable+Regulatory+Roles+in+Caenorhabditis+elegans.">An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans.</a> Science. 294 (5543), 858-862 (2001).</li><li>Yi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGCR8-dependent+microRNA+biogenesis+is+essential+for+skin+development.">DGCR8-dependent microRNA biogenesis is essential for skin development.</a> Proceedings of the National Academy of Sciences. 106 (2), 498-502 (2009).</li><li>Leung, A. K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+Ago2+binding+sites+from+mouse+embryonic+stem+cells+with+and+without+mature+microRNAs.">Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.</a> Nature Structural &amp; Molecular Biology. 18 (2), 237-244 (2011).</li></ol>

The authors declare no competing financial interests.

本文所描述的方法利用了几个关键变量最大化连接效率，即高浓度的PEG，用随机接头的，与高浓度的接头2,6,9的。这种方法可以从总RNA样品2可靠的定量测序文库。我们所进行的输入的RNA多步滴定，并得出结论认为，前面的方法是最适合的总RNA的量在1-8微克的范围（数据未显示）。当在10-500纳克范围的量使用，大部分的读取空间由适配器多联体，并从其中所述RNA连接酶被纯化细菌序列消耗。然而，值得注意的是在这些低输入实验是所述miR物种存在，虽然在低读出数，相对于相同的高输入样本仍然反射的实际数额。为了确保从总RNA样品中观察到的的miR型材是反射的实际数额，并且允许交叉询问不同的样品，我们通常包括三个合成的校准RNA的12 10倍稀释液。我们已经发现，包含0.1:0.01:0.001皮摩尔的鲜明校准RNA的产生有用读取号码，用于确认了测定的定量性质。 如在图1（见星号）表示合成的寡核苷酸通常包含异常分子是不同的，从有序序列大小。如果这些产品都能够共同净化与所述miRNA-3&#39;连接子混合，那么它可能是必要的PAGE纯化的3&#39;接头以下第M腺苷酸化。我们已经发现这是用于减少非特异性PCR产物的阴性对照样品中观察到的量有帮助的。 虽然上述方法进行了基准测试的miRNA的图书馆2定量的准备，我们充分预期，这是很容易的应用程序licable到更一般的程序，以及;即RNA测序和CLIP-SEQ（交联免疫沉淀）库的准备。实际上，我们已采用了一些前述方法的相同原理为CLIP-SEQ导致鲁棒文库的PCR相对于先前发表的报告13实验（数据未示出）。最后，血清miRNA的获得牵引力作为医疗诊断工具，我们预计前面的方法将鉴定的最强大和最可靠的miRNA的生物标志物的极大帮助。

高通量测序基础的方法已被广泛应用到许多生物样品近年来大大扩大我们的生物系统3,4的分子复杂性的理解。然而，制备RNA样品进行高通量测序的常赋予固有的就业方法的具体偏差，限制了这些强大的技术的潜在效用。这些具体的方法偏差得到了很好的证明了结扎为主，小RNA库准备1,2,5,6。这些偏见导致1000倍的变化读取号码等摩尔合成的miRNA，使得miRNA的丰推断，从测序数据疯狂可变且容易出错。 研究着重于噬菌体衍生的T4 RNA连接酶的性质已经证明，酶表现出的基于核苷酸的偏好7，它体现在高通量测序实验作为偏置库<sUP&gt; 1,2,8。为了最小化被RNA连接酶所赋予的偏见，多种策略已经使用;大分子拥挤9中 ，随机化是近端结扎部位6的适配器上的碱基序列，以及使用高浓度的结扎适配器2。通过这三种方法的组合，我们开发了一个工作流的小RNA文库的高通量测序（ 图1）兼容的无偏制剂。对于目前的协议和我们的优化方法进行直接的比较，请参阅最近的一份报告2。这种优化的方法产生的大于95％的在两个3&#39;和5&#39;步骤结扎的效率，并允许小分子RNA合成和生物样品2的无偏结扎。

<table><tbody><tr><td>3&#039; Linker (5&#039; phosphorylated, 3&#039; blocked)</td><td>Integrated DNA Technologies</td><td>custom</td><td></td></tr><tr><td>5&#039; Linker</td><td>Integrated DNA Technologies</td><td>custom</td><td>5&#039; blocked, HPLC purified</td></tr><tr><td>T4RNL2 (1-249 K227Q)</td><td>New England Biolabs</td><td>M0351S</td><td>Specialized for ligation of pre-adenylated DNA adapters</td></tr><tr><td>10x Ligation buffer (without ATP)</td><td>New England Biolabs</td><td>Included with M0351S</td><td></td></tr><tr><td>10x Ligation buffer (with ATP)</td><td>New England Biolabs</td><td>Included with M0204L</td><td></td></tr><tr><td>RNaseOUT</td><td>Invitrogen</td><td>10777-019</td><td></td></tr><tr><td>Polyethylene glycol (mol. Wt. 8,000)</td><td>New England Biolabs</td><td>Included with M0204L</td><td></td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9937</td><td>We have found water collected from a distillation apparatus to be of equvalent quality.</td></tr><tr><td>T4RNL1</td><td>New England Biolabs</td><td>M0204L</td><td></td></tr><tr><td>Superscript III RT kit</td><td>Invitrogen</td><td>18080-051&amp;nbsp;</td><td></td></tr><tr><td>Phusion PCR kit</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>Illumina RP1 primer</td><td>Integrated DNA Technologies</td><td>custom</td><td>Sequence information available from Illumina</td></tr><tr><td>Illumina RT primer</td><td>Integrated DNA Technologies</td><td>custom</td><td>Sequence information available from Illumina</td></tr><tr><td>Illumina index primer(s)</td><td>Integrated DNA Technologies</td><td>custom</td><td>Sequence information available from Illumina</td></tr><tr><td>40% Acrylamide</td><td>Fisher Scientific</td><td>BP14081</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U6504</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma Aldrich</td><td>A3678</td><td></td></tr><tr><td>Tetramethyethylenediamine (TEMED)</td><td>Sigma Aldrich</td><td>T9281</td><td></td></tr><tr><td>2x Denaturing RNA loading buffer</td><td>New England Biolabs</td><td>Included with M0351S</td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>SpinX Centricon tubes</td><td>Costar</td><td>CLS8161</td><td></td></tr><tr><td>Low retention microfuge tubes</td><td>Fisher Scientific</td><td>02-681-320</td><td></td></tr><tr><td>Sybr Gold</td><td>Invitrogen</td><td>S-11494</td><td></td></tr><tr><td>Adenylation kit</td><td>New England Biolabs</td><td>E2610L</td><td></td></tr></tbody></table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

miRNA的克隆和高通量测序，所谓的miR-SEQ，独树一帜的全基因组范围的方法来量化的miRNA与单核苷酸分辨率。这种技术捕获的miRNAs通过连接3&#39;和5&#39;寡核苷酸衔接头到miRNA分子，并允许从头 miRNA的发现。具有强大的新一代测序平台的耦合中，miR-SEQ一直在miRNA的生物学研究。然而，寡核苷酸连接的步骤介绍显著的偏见阻碍的miR-SEQ被用作一个精确的定量工具。以往的研究表明，在当前的miR-SEQ方法的偏见往往会导致不准确的miRNA定量与错误的高达1000倍的某些miRNA的1,2。要解决这些偏见，通过RNA结扎传授，我们已经开发出一种小RNA结扎方法导致95％以上的为3&#39;和5&#39;连接步骤连接效率。这个基准测试IM证明文库构建方法，使用等摩尔或差分混合的合成的miRNA，始终如一地产生具有与预期值小于2倍的偏差读出的数字。此外，这种高效率的miR-SEQ方法允许从体内总RNA样品2精确的全基因组miRNA表达谱。

预期的结果为前述方法最初应观测的DNA寡核苷酸，这是受腺苷酰化通过第M个 RNA连接酶（ 图2）的尺寸的单核苷酸移位（增加）的。下面的3&#39;连接，将丙烯酰胺凝胶的可视化表示（ 见图3）尖锐的高分子量条带在凝胶中的100-300核苷酸区域明显。这表明，总RNA样品使用是高品质（未劣化）。其次应该看到一个非常明亮的信号凝胶，这是多余的25个核苷酸区域，未连接3&#39;连接器。它也可能是有用的，包括在3&#39;连接一个没有输入的RNA控制。这将表明在3&#39;接头的纯度，并且还当非特异性信号变得有问题，可以通过进行与PCR步骤为的周期数表示。没有诊断为5&#39;连接在图4中示出但是是相同的1描述的进行了与放射性标记的RNA的反应表明，高浓度PEG8000所采用的重要性。最后，在PCR和非变性PAGE，146个碱基对的接头序列的从延长的PCR引物的大小，与miRNA插入和附加的序列一​​致的DNA产物可被观察到。要注意，如果PCR循环（这是根据经验确定）的适当数目被超过，则没有插入的miRNA产物可能掩盖所需的扩增子的大小是很重要的。 如图5所示，是从5皮摩尔合成的miRNA的，典型地为2微克总RNA所必需的PCR的13-18个周期的结果。 <img alt="图1" src="/files/ftp_upload/52095/52095fig1highres.jpg" /> 图1.原理图的工作流程2步结扎小RNA文库制备的:Shown是准备一个不带偏见的miR-SEQ库的一般步骤。以黑色示出是3&#39;DNA连接适配器，它是通过腺苷酸化在步骤1中激活，miRNA的显示为绿色，并连接到3&#39;的适配器在步骤2中，和5&#39;核糖核酸结扎适配器是在蓝色被连接到所述在步骤3中的编号步骤嵌合分子中详细的协议部分中描述，斜体表示酶促步骤。 <img alt="图2" src="/files/ftp_upload/52095/52095fig2highres.jpg" /> 图2的3腺苷酸化&#39;。连接器与第M的RNA连接酶的DNA寡核苷酸的腺苷酸化的18％PAGE-尿素凝胶以用于随后的3&#39;连接反应，（+）表示样品被孵育第M RNA连接酶，（ - ）表示样品温育不含酶，和（m）表示的大小的标准（示号码一吨右中碱基对）。在左边的是寡核苷酸的物种存在的示意图。星号表示了异常的寡核苷酸合成产生较大的DNA的产品。 <img alt="图3" src="/files/ftp_upload/52095/52095fig3highres.jpg" /> 图3.合成和总RNA样品3&#39;结扎。 A）的 15％PAGE-尿素3凝胶&#39;连接反应，（μm）表示的大小的标准（在左侧的数字表示碱基对），（无RNA）的表示无输入RNA和3的反应"连接体，这不是凝胶纯化，（+）表示以5pmol的合成的miRNA和3进行结扎&#39;连接基为凝胶纯化，（2和1μg）表示进行3小鼠的总RNA的量&#39;结扎用相同的，凝胶纯化的连接物，星号表示与P 32进行连接反应超出3&#39;3相似的连接器。B）放射自显影"<html> 5&#39;末端标记合成的miRNA。在上面的数字表示时间（hr）使反应继续进行，并且在底部显示连接为未连接的和连接的总和的百分比的量。 <img alt="图4" src="/files/ftp_upload/52095/52095fig4highres.jpg" /> 图4. 5&#39;的放射性标记的miRNA-3&#39;Linker杂交结扎。5放射自显影&#39;连接反应进行以P 32的5&#39;端标记的合成的miRNA-3&#39;连接子混合。在上面的数字表示的PEG的反应中使用量，并且在底部的数字表示连接为未连接的和连接的总和的百分比的量。线在左侧的是连接分子的示意图，其中miRNA是被显示为绿色，5&#39;和3&#39;接头分别是蓝色和黑色。 PLOAD / 52095 / 52095fig5highres.jpg"/&gt; 图5. PCR产生小RNA的DNA文库。8％纯从的miR-SEQ结扎过程中产生的PCR产物的PAGE。在数字上面显示的PCR循环，在一侧的数字表示分子大小的标准。从各PCR用DNA种类被确定在右边。格子图案是由于自体荧光样品菜。

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

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

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 ...

highly-efficient-ligation-small-rna-molecules-for-microrna

Universidad San Sebastian

Research

JoVE Journal

Biology

11.8K 次观看.  University of Colorado, Boulder. 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. 

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

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

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 ...

遗传学

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

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

使用 AQRNA-seq 分析 tRNA 动力学

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

作者聚焦:AQRNA-seq 在绘制小 RNA 和解开蛋白质翻译机制中的作用

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

用于定量小 RNA 的 AQRNA-seq

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

发育生物学

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

小RNA分子的高效结扎的微小RNA定量高通量测序

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 Fluidigm公司动态ArrayTM国际金融中心

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

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

用于植物微RNA检测和定量的RNA斑点分析

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

为早期小鼠胚胎测序准备小型RNA库

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

小RNA分子的高效结扎的微小RNA定量高通量测序

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

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

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

用于植物微RNA检测和定量的RNA斑点分析

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

为早期小鼠胚胎测序准备小型RNA库

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

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