这项工作得到了E.S的IMPS博士津贴、EMBO对V.P.的长期研究金和对F.K.的MPI-MPP内部资金的支持。 这项工作的一部分也通过PLAMORF提供资金，该项目已获得欧洲研究理事会（ERC）根据欧洲联盟的Horizan 2020研究与创新方案（赠款协议第810131号）提供资金。作者要感谢费德里科·阿佩尔特对手稿的生物信息分析和评论，以及马蒂厄·巴欣和阿米拉·克拉姆迪的生物信息分析。

1. 准备RNA
<ol><li>将200毫克的植物组织研磨到液氮粉末中，确保组织在整个过程中保持冷冻。</li>
	<li>按照硫酸-苯酚-氯仿提取方案，从所需的植物组织中提取RNA。为了减少在相分离过程中用DNA污染RNA的可能性，请使用1-溴-3-氯丙烷代替氯仿。<ol>
<li>将含有瓜尼丁硫酸酸和苯酚酸的RNA提取试剂加入研磨植物组织（每100毫克组织500μL）。通过倒置混合好，并确保所有的组织都是湿的。在室温下孵化10分钟，分离核糖核蛋白复合物。</li>
		<li>离心机10分钟，在12，000 x g 在4°C，并将超纳坦转移到一个新的1.5 mL管。</li>
		<li>加入 200 μL 的 1-bromo-3-氯丙烷（每 500 μL RNA 提取试剂 100 μL）和涡流。</li>
		<li>离心机在 4 °C 下在 12，000 x g 下旋转 15 分钟，并将上水相（约 500 μL）转移到新的 1.5 mL 管中。</li>
		<li>加入 1 卷异丙酚 （500μL） 和 0.1 卷 3 M 醋酸钠 pH 5.5 （50 μL），在 -20 °C 下倒置和沉淀 10 分钟，混合均好。 注:建议使用醋酸钠（NaOAc），以增强RNA降水。此时，通过将RNA的降水时间延长数小时甚至一夜之间，可以暂停协议。</li>
		<li>离心机 30 分钟， 在 12，000 x g 在 4 °C 和丢弃超纳坦。</li>
		<li>用 80% EtOH 的 500 μL 清洗颗粒两次，在 4 °C 下在 12，000 x g 下将离心机洗涤 5 分钟，然后丢弃。</li>
		<li>用 500 μL 99% EtOH 清洗颗粒一次，离心机在 4 °C 下在 12，000 x g 下清洗 5 分钟，然后丢弃。</li>
		<li>干燥颗粒5-10分钟，溶解在30μL无RNase H2O。 注:相反，使用任何选择的RNA提取协议（例如，基于列的系统）。如果协议中包含 DNase 消化，请在以下步骤中跳过它（步骤 1.3）。</li>
	</ol></li>
	<li>测量RNA浓度（例如，使用光谱仪），并用DNase消化20μg的RNA。 注:DNA富含m5C，抗体不区分DNA和RNA。<ol>
<li>在典型的 DNase 反应中，在 50 μL 反应中处理 10μg 的RNA。混合以下组件，在 37 °C 下孵化反应 30 分钟: 10μg 的RNA x μL 10x 稀会缓冲区 5 μL D纳塞 1 μL （2 单位） 无鼻 H2O 高达 50μL</li>
		<li>通过添加适量的 DNase 灭活试剂（如果该试剂盒包含在 DNase 套件中并根据制造商的说明），或执行清理步骤（例如柱净化或苯酚/氯仿萃取），去除酶。</li>
	</ol></li>
	<li>检查毛细纤维电泳分离RNA的质量和纯度，如果RNA完整性数字（RIN）高于7，则继续检查，以确保样品质量良好。</li>
	<li>可选:删除核糖体RNA，以丰富mRNA含量的样品使用rRNA去除套件，并根据制造商的协议。<ol>
<li>使用前一步骤的 DNase 处理 RNA 进行 rRNA 耗竭反应。如果总RNA的量超过反应建议的最大量，则执行多个反应。</li>
		<li>请注意，只有 5-10% 的输入量将在 rRNA 耗尽后恢复。继续使用RNA耗尽RNA（所有样品的等量），忽略以下步骤中提到的金额，因为它们指的是总RNA。 注:有关现有RNA耗竭方法的比较和描述，请参阅参考文献38、39、40。rRNA 是总RNA的主要部分，在许多生物体中甲基化为m5C。</li>
	</ol></li>
	<li>提前准备体外成绩单 （IVT）， 用作对照 RNA 序列，并将其添加到样本中。<ol>
<li>使用体外转录套件生产两种不同的 IVT，一种是非甲基化核苷，另一种是 rCTP 被 5-甲基-rCTP 取代，分别用作 MeRIP 中的负对照和正对照。编写的抄本是 埃格夫普 和 雷尼拉· 卢西费拉塞的笔录。 注:IVT不应存在于您正在分析的生物体的转录体中。如果 IVT 来自同一个模板（例如，两个 EGFP），则在两个不同的样本中添加正负对照。如果他们的序列不同（例如，EGFP和雷尼拉），它们可以添加到同一个样本中。</li>
		<li>作为对照组，每个样本中每 3 μg RNA 的峰值为 0.1 ng IVT。</li>
	</ol></li>
	<li>将RNA声波化为大约100nt片段。 注:RNA剪切的条件必须提前调整，每个声波器的条件也不同。对于此处使用的模型，声波处理具有以下条件:峰值功率 174、负载因子 10、循环/爆裂 200、17 分钟。<ol>
<li>所有样品的RNA量相同，在总体积的80μL（最小60微升，最大100微升）中，每个样品至少12μg RNA，填充无RNase H 2 O。</li>
	</ol></li>
	<li>通过毛细纤维化确认声波效率和RNA样品浓度。碎片RNA的平均大小应在100 nt左右。</li>
</ol>2. 甲基化RNA免疫沉淀（梅里普）
<ol><li>在低绑定管中，添加 9 μg 的声波RNA 和无 RNase H2O 高达 60 μL（或更多，具体取决于浓度）。</li>
	<li>在水浴中以70°C加热RNA10分钟，在冰水中再冷却10分钟，从而分离二级结构。</li>
	<li>将样品分成三部分:三分之一（20 μL，如果在步骤 2.1 中取了 60 μL）保存在单独的管子中，以 -80 °C 作为输入样本。将剩余的 40 μL 填充无 Rnase H2O 至 860 μL，然后分成两个低绑定管:一个用于 IP，另一个用于模拟控制（每个管 430μL）。</li>
	<li>添加到两个管子: 50μL 的 10 倍梅里普缓冲区 10μL 的 RNase 抑制剂 模拟样品中每 10 μL H2O 的 IP 样本中的 10 μl α-m5C 抗体 （10 微克） 注:以前使用的抗体克隆不再在商业上使用。然而，任何抗5-甲基西氨酸单克隆抗体都应该同样起作用。抗体应测试的特异性之前，用于梅里普11，23。</li>
	<li>用准胶片密封管子，在4°C下孵育12-14小时，头顶旋转。</li>
	<li>第二天，准备蛋白质G磁珠结合。<ol>
<li>对于每个管（IP 或模拟控制），使用 40μL 的珠子。添加珠子总数（#管x 40μL， 例如，对于 2 个 IP 和 2 个模拟样品，需要 160μL 的珠子）在 15 mL 管中，用每个样品 800μL 的 1 倍 MeRIP 缓冲器洗三次（#管 x 800 μL 缓冲区，例如，2 个 IP 和 2 个模拟样品需要 3.2 mL）。</li>
		<li>在室温下进行5分钟的头顶旋转清洗，在磁架的帮助下收集珠子，并丢弃洗涤缓冲区。第三次清洗后，将珠子重新悬在与采集的珠子初始体积相同的 1 倍 MeRIP 缓冲区中（管 x 40 μL 的#，例如，160μL 的 1x MeRIP 缓冲区，用于 2 个 IP 和 2 个模拟样品）。 注:所使用的蛋白质G珠的量由特定抗体类型的珠子的结合能力和所使用的抗体量决定。在这种情况下，珠子的结合能力为每毫克珠子约8μg的鼠标IgG和30毫克/mL浓度。因此，40 μL 足以结合约 9.6 μg 的抗体。</li>
	</ol>
</li>
	<li>在每个 IP 和 Mock 样本中添加 40 μL 的重新悬生珠子，并在 4 °C 下孵育 2 小时，并进行开销旋转。</li>
	<li>将管子放在磁架上 1 分钟，丢弃超纳坦或将其保存为对照（非绑定 RNA 样本）。</li>
	<li>通过在 1x MeRIP 缓冲器的 700 μL 中重新悬念 5 次，用 0.01% Tween 20 进行冲洗珠子，并在室温下通过头顶旋转孵育 10 分钟。</li>
	<li>在 200 μL 蛋白酶 K 消化缓冲液中重新悬生洗过的珠子，并在 50 °C 下加入 3.5 μL 蛋白酶 K. 孵化剂 3 小时，在 800 rpm 时摇晃。偶尔，如果在孵化过程中形成珠子沉积物，则手动轻拂管底。</li>
	<li>通过添加 800 μL 的RNA 提取试剂并遵循硫酸钛酸-苯酚-氯仿提取方案提取RNA，并按步骤 1.2 继续。为了提高RNA颗粒的能见度，在降水步骤中可以在异丙醇中加入彩色共沉淀剂。在无 RNaseH2 O 的 20 μL 中重新悬念颗粒（或等于第 2.3 步保持的输入体积）。</li>
</ol>3. 下游分析
<ol><li>提交输入和 IP 样本，进行单端测序，50 个碱基读取长度 （SE50）。</li>
	<li>使用切口41 修剪 3' 端适配器，丢弃小于 48 nt 的读数。</li>
	<li>使用 STAR42 对阿拉比多普西斯基因组 （TAIR10 注释） 进行地图修剪读取，因不匹配和最大内子大小为 10 kb 而截止 6%。保持唯一映射读数以供进一步分析。</li>
	<li>使用两种不同的方法识别 IP 样本中丰富的 RNA 片段与输入中的丰富RNA 片段，并考虑发现两者都显著丰富的片段。<ol>
<li>首先，使用 MACS2 峰值呼叫者43 在汇集的 IPs 与输入上检测 MeRIP-seq 峰值。</li>
		<li>其次，按照迈耶等人22 号和杨等人17号描述的梅里普-塞克峰呼叫分析。<ol>
<li>使用自定义 R 脚本，将基因组分成不同的 25 nt 窗口，并根据上次映射的核苷酸的位置（因为读数源自 100 nt RNA 片段）计算每个窗口唯一映射读数。</li>
			<li>与费舍尔精确测试的输入相比， 计算 IP 样本中的明显丰富窗口。使用本杰明尼 - 霍奇伯格程序纠正多个测试。</li>
			<li>保留至少连续两个窗口和仅覆盖一个窗口的废弃山峰的显著丰富山峰。</li>
		</ol>
</li>
	</ol>
</li>
	<li>通过这两种方法发现具有显著丰富峰值的基因组（成绩单）的注释区域。</li>
	<li>或者，或补充，测试特定的RNA目标，以丰富其在IP样本。<ol>
<li>反向转录RNA（输入、IP和模拟样本的相同体积）与随机六角形。</li>
		<li>对所选目标执行定量实时 PCR，通过ΔΔCt方法比较输入、IP 和模拟。 注:生成的产品不应长于 100 bp，因为这是平均碎片大小。</li>
	</ol>
</li>
</ol>

作者没有利益冲突可以披露。

RNA携带超过一百个不同的基础修改4，形成表征44。这些修改增加了一层对翻译和信号的监管（在5、6、8、20、45、46中审查）。早期的研究能够检测RNA28，47的转录后修改的存在，但具体的修改RNA需要确定，以了解表皮学的作用。MeRIP被设计成一种绘制RNA甲基化位点图的方法，全谱21，22。它可以适应任何修改，如果一个特定的抗体可用。
此协议的主要优点是，对于RNA和用户来说相对简单、安全（例如，5-azaC对植物和人类具有剧毒），不需要序列或修改酶信息。此外，与不包含浓缩步骤的双硫酸盐测序不同，IP 对甲基化RNA的浓缩增加了检测低丰度 mRNA 的机会。当执行两轮连续的梅里普时，含有甲基化位点的RNA片段的浓缩量进一步增加22。MeRIP的局限性之一，特别是应用于mRNA甲基化研究时，是作为检测输入所需的大量RNA。核糖核酸消耗（或聚（A）浓缩）步骤将减少严重改性核糖体RNA造成的背景，但它可去除总RNA的90%以上。DNA也必须完全去除，因为它富含5甲基化细胞素。另一个缺点是甲基化精确位置的分辨率较低。用抗体孵化前RNA的声波有助于朝这个方向发展，将含有改性剂的区域缩小到100-200核苷酸。当 MeRIP 与深度测序相结合时，m5C 站点预测的分辨率会随着测序读数形成围绕潜在甲基化站点的高斯分布而增加。此外，抗体的特异性需要在检测前得到确认（例如，用RNA点斑点检测，用改性核苷酸合成的寡头进行），然而，抗体在多大程度上实际上可以区分密切相关的修饰（例如，m6A 和 m6Am）是该领域48、49中的一个争论点。此外，高度结构化的RNA可能会干扰抗体-抗原相互作用，另一个限制主要通过在IP之前RNA的分裂和自然来解决。相反，同样受二次结构影响的双硫酸盐测序不包括碎片步骤，这可能是造成m5C位点与双硫酸盐测序16和MeRIP-seq11、17预测的mRNA之间差异的原因之一。其他细胞氨酸修饰（例如，hm5C）也对双硫酸盐介质脱氨35具有抗药性。
MeRIP-seq 的修改包括交叉链接步骤， 要么引入光活性核糖核苷酸（光交叉链接辅助m6A-seq，PA-m6A-seq 50），要么使用紫外线在IP（miCLIP49，不同于导言30中描述的miCLIP，但也与单核苷酸分辨率不同）后创建抗体-RNA交叉链路。将来，随着有关RNA甲基化的知识的积累，基于甲基化出现的修改酶和/或协商一致的序列，更有针对性的方法可能更可取。读取蛋白的识别对于理解转录后修改的分子和信号功能至关重要。Nanopore 测序技术已经允许在没有事先处理 RNA17 的情况下直接识别改性核苷酸，但在序列深度和生物信息分析方面仍有改进的余地。总体而言，MeRIP-seq 目前是识别甲基化RNA成绩单的既定、可靠和公正的方法。

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/61231">Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis</a>. The author list was updated.
The author list was updated from:
Eleftheria Saplaoura1, Valentina Perrera2, Friedrich Kragler1 
1Max Planck Institute of Molecular Plant Physiology 
2Department of Molecular Medicine, Medical School, University of Padua
to:
Eleftheria Saplaoura1, Valentina Perrera2, Vincent Colot3, Friedrich Kragler1 
1Max Planck Institute of Molecular Plant Physiology 
2Department of Molecular Medicine, Medical School, University of Padua 
3Biologie de l&#39;Ecole Normale Sup&#233;rieure (IBENS),&#160;Paris, France

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/61231">Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis</a>. The author list was updated.

Erratum: Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

在所有三个生命王国中，RNA物种都经过了转录后修改，对这些功能相关的生化修饰的研究被称为"表征学"。Epitranscript经济学是一个不断增长的领域，正在开发各种方法来研究和绘制RNA分子的修饰图（在1，2中回顾）。已发现一百多个RNA修改，检测到在rRNA，tRNA，其他ncRNA，以及mRNA3，4。虽然在tRNA和rNA中化学多样化的转录后修改的存在和功能被广泛研究5，6，7，8，直到最近才有mRNA修改的特点。在植物中，许多mRNA修改已被确定至今，包括m7G在帽结构9，m1A10，hm5C11，12，和尿化13。然而，只有米6A10，14，15，米5C11，16，17，和伪尿丁18被映射在阿拉比多普西斯全转录。后转录mRNA基地修改涉及几个发展过程19，20。
表皮经济学中最常用的方法之一是甲基化RNA免疫沉淀，以及深度测序（MeRIP-seq）。MeRIP-seq于2012年开发，以研究哺乳动物细胞21，22的m6A。它需要使用抗体进行所需的修改，旨在丰富携带改性核苷酸的RNA片段。之后通常进行深度测序，以识别和映射丰富的碎片或定量 PCR 以验证特定的 RNA 目标。MeRIP 的准确性基于抗体的特异性，以识别经过类似修改的改性核苷酸（例如，m 5 C 和 hm5C11，23）。除了m6A外，MeRIP-seq还应用于研究11、17、23、24、25等多种生物体的m1A和m5CRNA甲基化。
细胞素在第五碳位（m5C）的甲基化是最普遍的DNA修饰26，27和最常见的RNA修饰之一太3，4。虽然m5C在1975年28年在真核mRNA中被发现，但最近才有研究重点绘制修改转录全，在编码和非编码RNA11，16，17，23，29，30，31，32，33，34。
m5C RNA 研究中使用的替代方法包括将非甲基化细胞氨酸化学转化为尿素（双硫化物测序）和基于已知RNA细胞氨酸甲基转移酶与其RNA靶点不可逆转的结合的免疫沉淀检测（miCLIP，aza-IP）。简言之，双硫化物测序利用了5甲基化细胞素的特性，对二硫化钠治疗具有抗药性，这种治疗将未修饰的细胞素去除尿素。该方法最初是为DNA开发的，但也适用于RNA，许多研究都选择了这种方法来检测RNA16、23、29、32、34、35中的m 5C位点。miCLIP 和 aza-IP 都需要以前对 RNA 细胞氨酸甲基转移酶的了解和各自抗体的使用。在miCLIP（甲基化单核苷酸分辨率交叉链接和免疫沉淀）的情况下，甲基转移酶携带单个氨基酸突变，使其与RNA基板结合，但不能释放30。在 aza-IP （5-阿扎西丁 - 中介RNA免疫预测） 中，当 RNA 聚合酶由 RNA 聚合酶结合到目标 RNA 分子31时，5-azaC 核苷和 RNA 细胞素甲基转移酶之间形成不可逆转的结合。
这三种方法的主要优点是允许单核苷酸分辨率映射m5C。此外，miCLIP 和 aza-IP 提供有关选定的 RNA 细胞氨酸甲基转移酶的具体目标的信息，深入破译转录后 RNA 修改的机制和作用。但是，MeRIP-seq 方法可以在不需要任何事先知识的情况下识别全转录体 m5C 区域，并避免苛刻的化学和温度条件，例如双硫酸盐处理或 5-azaC 的孵化。梅里普和双硫酸盐测序都可以通过二级RNA结构36抑制。免疫沉淀前 MeRIP 检测中包含的碎片步骤旨在促进抗体结合并增加 m5C 识别的分辨率。
另一个值得一提的方法是RNA核苷的质谱（MS）。MS 可以在 DNA 和 RNA 上检测和区分任何类型的修饰。简言之，RNA被提取和DNase消化，然后脱盐和消化到单核苷酸。RNA核苷由质谱仪分析。这种方法可用于量化每个修改的水平，它不依赖于抗体或化学转换。然而，一个主要缺点是，它提供了大量有关RNA修改存在的信息。为了绘制修改图，MS 需要与 RNase 消化和特定RNA分子的测序信息相结合，例如人类 tRNALeu（CAA）37。
在这里，我们描述和讨论梅里普测定用于研究m5C RNA甲基化在阿拉比多普西斯17。

<table><tbody><tr><td>anti-m&lt;sup&gt;5&lt;/sup&gt;C antibody</td><td>ZymoResearch</td><td>A3001 (discontinued), A3002 available</td><td>Clone 10G4 (discontinued), 7D21 available</td></tr><tr><td>Chip and reagents for capillary electrophoresis</td><td>Agilent</td><td>5067-1511</td><td>RNA 6000 Nano kit</td></tr><tr><td>Dnase</td><td>Ambion</td><td>AM1907</td><td>TURBO DNA-free kit</td></tr><tr><td>Co-precipitant of nucleic acids</td><td>Ambion</td><td>AM9515</td><td>Glycoblue, 15 mg/mL</td></tr><tr><td>&lt;em&gt;In vitro&lt;/em&gt; transcription kit</td><td>Promega</td><td>P1300</td><td>T7 RiboMAX Large Scale RNA Production System</td></tr><tr><td>Low-binding reaction tubes</td><td>Kisker Biotech</td><td>G017</td><td>1.7 ml microcentrifuge tubes</td></tr><tr><td>Protein G magnetic beads</td><td>Invitrogen</td><td>10003D</td><td>Dynabeads Protein G</td></tr><tr><td>Proteinase K</td><td>Ambion</td><td>AM2546</td><td>20mg/mL stock solution, RNA grade</td></tr><tr><td>RNase inhibitor</td><td>Promega</td><td>N2615</td><td>RNasin Plus 40 U/&amp;mu;l</td></tr><tr><td>rRNA removal kit</td><td>Epicentre</td><td>RZPL11016</td><td>Ribo-Zero rRNA removal kit (Plant leaf)</td></tr><tr><td>Sonication tubes</td><td>Covaris</td><td>520045</td><td>microtube AFA Fiber Pre-Slit Snap-Cap 6x16mm</td></tr><tr><td>RNA extraction reagent</td><td>Ambion</td><td>15596018</td><td>TRIzol reagent</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Capillary electrophoresis machine</td><td>Agilent</td><td>G2939BA</td><td>2100 Bioanalyzer System</td></tr><tr><td>Magnetic rack</td><td>Invitrogen</td><td>12321D</td><td>DynaMag-2</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>discontinued</td><td>5417R model</td></tr><tr><td>Rotator</td><td>Benchmark</td><td>R4040</td><td>RotoBot Mini</td></tr><tr><td>Sonicator</td><td>Covaris</td><td>500217</td><td>S220 Focused-ultrasonicator</td></tr><tr><td>Spectrophotometer</td><td>ThermoFisher Scientific</td><td>ND-ONEC-W</td><td>NanoDrop OneC</td></tr><tr><td>Waterbath</td><td>GFL</td><td>1012</td><td>1012 Incubation bath</td></tr></tbody></table>

methylated rna immunoprecipitation assay to study m5c modification in arabidopsis

RNA上的二次碱基修饰（如m5C）会影响改性RNA分子的结构和功能。甲基化RNA免疫沉淀和测序（MeRIP-seq）是一种旨在丰富甲基化RNA并最终识别修改后成绩单的方法。简言之，声波化RNA与5甲基化细胞素的抗体一起孵育，并在蛋白质G珠的帮助下沉淀。然后对浓缩碎片进行测序，并根据读数分布和峰值检测绘制潜在的甲基化位点。MeRIP可以应用于任何有机体，因为它不需要任何先前的序列或修改酶知识。此外，除了碎片外，RNA不接受任何其他化学或温度处理。然而，MeRIP-seq不像其他方法那样提供甲基化位点的单核苷酸预测，尽管甲基化区域可以缩小到几个核苷酸。使用不同的改性特异性抗体，可以根据RNA上存在的不同碱基修饰对MeRIP进行调整，从而扩展此方法的可能应用。

图1提供了该方法的示意图。协议的第一个关键步骤是获得质量良好的RNA（RIN ≥ 7），并将其声波化到大约 100 nt 片段。两个步骤的效率都由芯片毛细细的电泳机检查。在图 2A中，显示了一个好的RNA样本的代表性运行。样品在装载到芯片上之前被稀释 1:10，以便浓度在所用套件检测范围 （5-500 ng/μL） 中。同一样本也在声波后运行，并显示在图2B 中。请注意，图左侧有一个统一的峰值，大小约为 100 个核苷酸。浓度降低既是由于碎片过程中RNA损失造成的，也是由于样品体积增加（60-100 μL，步骤1.7.1）。
IP 和模拟样品的质量可以通过 qRT-PCR 进行评估。为此，尖峰IVT充当正负对照:甲基化IVT，在所有细胞素位置有m5C，预计将在IP样本中高度丰富:相反，非甲基化试管婴儿不应该有IP和模拟之间的区别。用于两个控制 IVT（EGFP 和 雷尼拉 卢西费拉斯） 的 qRT-PCR 检测的引物列在 表 1中。事实上，如 图3所示，大约80%的甲基化IVT是在IP样本中恢复的，而在模拟中只有大约2%。对于非甲基化控制，IP 和模拟样本的恢复率均低于 1%。这验证了 MeRIP 的效率，即甲基化 RNA 片段沉淀和丰富，并且是样品可用于下游分析的良好指标。此外，非甲基化 IVT（IP 与模拟比）的折叠浓缩可用作估计 qRT-PCR 检测中浓缩意义的阈值。
将读数对齐到基因组（图4）后，应用步骤 3.4.1 和 3.4.2 中描述的峰值调用算法来识别具有统计学意义的窗口，与输入相比，IP 样本中的内容丰富。与这些窗口相对应的序列可以进一步使用，例如搜索保存的甲基化相关图案11，17。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61231/61231fig01.jpg"> 图1:梅里普-塞克协议的示意图表示。 RNA样本用5-甲基化细胞氨酸的抗体孵化，复合物用蛋白质G磁珠拉下，与绑定RNA一起捕获抗体。通过深度测序和 qRT-PCR 分析已分离的 RNA 样本。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61231/61231fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61231/61231fig02.jpg"> 图2:RNA样品质量分析的代表性结果。 （A） 合格总RNA工厂样本的代表性概况。（B） 声波化后RNA样本的代表性配置文件至100nt片段。毛细纤维电泳软件的输出文件。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61231/61231fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61231/61231fig03.jpg"> 图3:控制IVT的qRT-PCR分析。 甲基化和非甲基化体外记录分别用作 MeRIP 检测的正对照和负对照。免疫沉淀后，甲基化IVT在IP样品（绿色）中高度丰富，但在模拟样品中（无抗m5C抗体;紫色）。非甲基化 IVT 显示没有丰富，IP 和模拟之间没有区别。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61231/61231fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61231/61231fig04.jpg"> 图 4: 阅读 MeRIP 前后的对齐，围绕有代表性的脚本。 读取与输入示例（上行）中的特定成绩单对齐，并在两个 IP 副本（中间和下行）中读取。黑匣子显示一个根据 MACS243 和 MeRIP-seq22 峰值调用分析识别的丰富 50 nt 长窗口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61231/61231fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>目标</td>
			<td colspan="2">引子对</td>
			<td colspan="2">产品序列</td>
			<td>产品长度</td>
		</tr>
<tr>
<td rowspan="2">埃格夫普</td>
			<td colspan="2">对于: 5'- 格卡塔卡加克 - 3'</td>
			<td colspan="2" rowspan="2">格卡塔卡加加格 格特加卡茨格 格特加奇卡格特加格茨加格茨</td>
			<td rowspan="2">72 bp</td>
		</tr>
<tr>
<td colspan="2">修订: 5'- 格茨卡格特加特 -3'</td>
		</tr>
<tr>
<td rowspan="2">雷尼拉·卢西费拉斯</td>
			<td colspan="2">对于: 5'- 加加塔特克特加特加加 - 3'</td>
			<td colspan="2" rowspan="2">加加塔特克特加加克 阿特格卡特卡塔加 阿格塔加卡加加特加特加茨</td>
			<td rowspan="2">75 bp</td>
		</tr>
<tr>
<td colspan="2">修订: 5'- 格茨卡特克塔 -3'</td>
		</tr>
</tbody></table>表1:有关qRT-PCR分析的引物和生成产品的信息。
缓冲食谱。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61231/Buffer%20Recipes%20MeRIP.xlsx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis at JoVE.com

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

甲基化RNA免疫沉淀检测是一种基于抗体的方法，用于丰富甲基化RNA片段。再加上深度测序，它导致识别承载m5C修改的抄本。

甲基化RNA免疫沉淀分析研究m5C在阿拉伯的修改

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

methylated-rna-immunoprecipitation-assay-to-study-m5c-modification

Nanyang Technological University

Research

JoVE Journal

Genetics

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

6.5K 次观看.  Max Planck Institute of Molecular Plant Physiology. 甲基化RNA免疫沉淀检测是一种基于抗体的方法，用于丰富甲基化RNA片段。再加上深度测序，它导致识别承载m5C修改的抄本。

视频: Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

科研

生物化学

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

遗传学

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA甲基转移酶活性分析的无抗体测定

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

发育生物学

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

使用质谱法表征单个神经元中的RNA修饰

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

化学

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

生物学

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

神经科学

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

探索病毒感染时的m6A和m5C表继转录组:HIV示例

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

甲基化RNA免疫沉淀分析研究m^5C在阿拉伯的修改

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一种测量方法RNA