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/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/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>&#945;&#8208;m5C 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>In vitro 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/&#956;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>Equipment</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>Microentrifuge</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/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/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/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/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/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

Research

JoVE Journal

Genetics

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

6.4K 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

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...