C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).

注:总RNA米6 A级包括rRNA基因，基因，和其他小RNA。由于核糖体RNA具有丰富的米6一修改，m的测量6 A水平将需要考虑这一事实。 1. RNA分离<ol><li>使用核糖核酸酶净化解决方案（喷在纸巾），擦拭移液器和实验室工作台表面。无核酸酶水的湿纸巾，再次擦拭移液器和实验室工作台表面。 </li><li> RNA提取<ol><li>总RNA分离<ol><li>用顶置式搅拌器，均化50-100毫克组织或5-10×10 6个细胞在1mL的RNA分离溶液保持在4℃。 注:更多组织（100-150毫克）可能需要从因为较低的RNA浓度在其中的小鼠脂肪组织样品。样品从小鼠心脏，肝脏，骨骼肌，肺，脑来源，和巨噬细胞先前已经采用4。 </li><li>在cubate样品匀浆在室温下5分钟。 </li><li>添加100μL的每1毫升样品匀浆1-溴-3-氯丙烷（BCP）的。摇晃试管剧烈15秒，然后继续根据制造商的说明24总RNA分离。 </li></ol></li><li>从分离的总RNA多腺苷酸mRNA纯化<ol><li>净化用可获得的试剂盒或协议多腺苷酸的mRNA。 <ol><li>摇彻底重新暂停各通过以下方法解决:2倍的结合溶液，寡（dT）的聚苯乙烯珠和洗涤溶液。确保寡（dT）在使用前珠温至室温。 </li><li>传输的每制备洗脱溶液120微升成管并加热至70℃的加热块中。 </li><li>吸取到的总RNA的500微克到离心管中。无核酸酶的水，将音量调节到250微升。 </li><li>添加的2倍结合液250微升总RNA所以lution和涡简单地混合内容。 </li><li>添加低聚15μL（DT）珠和涡彻底混合。 </li><li>孵育在70℃下该混合物3分钟。 </li><li>除去来自加热块的样品，并让它在室温下放置10分钟。 </li><li>离心在15,000rpm xg离心2分钟。 </li><li>小心取出上清液，留下约50微升。 </li><li>加的洗涤液500微升重新悬浮，通过移液沉淀。 </li><li>移液器悬挂到自旋过滤/收集管套。 </li><li>离心在15,000rpm xg离心2分钟。除去从收集管色谱柱和丢弃流过。返回列至收集管。 </li><li>吸取500微升洗涤液到旋转过滤器。 </li><li>离心15,000× 克2分钟。 </li><li>转移自旋过滤到一个新的收集管。 </li><li>移液器50的洗脱液μL加热至70℃到旋转过滤器的中心。 </li><li>在70℃孵育2-5分钟。离心在15,000rpm xg离心1分钟。 </li><li>吸管至70℃洗脱溶液加热额外的50微升到旋转过滤器的中心。 </li><li>重复步骤1.2.2.1.18。 </li></ol></li><li>加入100微克/毫升糖原，0.1体积的3M乙酸钠缓冲液（pH 5.2）和2.5体积的无水乙醇中。在-80℃沉淀过夜。 </li><li>离心机以15,000 xg离心在4℃下25分钟。弃去上清液。 </li><li>用1mL的75％乙醇洗涤沉淀。离心机以15,000 xg离心在4℃下15分钟。小心地取出乙醇。 </li><li>干燥空气中为3-5分钟。 </li><li>加的不含核酸酶的水10μL的以溶解沉淀。 </li><li>储存于-70℃。 </li></ol></li></ol></li><li> RNA定性和定量<ol><li>使用分光光度计，确定由NotI位RNA浓度纳克在260nm和280nm的吸光度。在A 260 / A 280的比例应为1.8-2.2。 </li><li>检查用0.8％琼脂糖凝胶电泳25的RNA样品的质量。 注:完整的真核细胞的总RNA应该表现出强烈的28S和18S rRNA的乐队。 28S的18S与rRNA基因带强度有一个良好的RNA制备的比率是〜2。 </li></ol></li></ol> 2.凝胶电泳和转让注:缓冲液配制协议在表1中给出。 <ol><li>甲醛凝胶制备（1％） <ol><li>冲洗所有电泳设备酸二乙酯（DEPC）水。 </li><li>熔融2.5克在215毫升DEPC水的琼脂糖完全在微波炉。 </li><li>加12.5毫升10×3-（N-吗啉代）丙磺酸（MOPS）缓冲液和22.5毫升37％的甲醛。 </li><li>倒入用1.5毫米厚的梳子的电泳装置。 </li><li>消除泡沫或推Ť下摆凝胶用干净的梳子的边缘。 </li><li>使凝胶在室温下固化。 </li></ol></li><li>样品制备<ol><li>混合样品缓冲液中的1-10微克总RNA和11.3微升。 </li><li>混合RNA标记（1微克/微升）的2微克的样品缓冲液11.3微升。 </li><li>不含核酸酶的水从2.2.1和2.2.2添加到样本以16微升的总体积。 </li><li>热，在60℃进行5分钟，然后在冰上冷却。 </li><li>混合来自2.2.3样品16微升与4微升跟踪染料，其含有0.1微克/微升的溴化乙锭在冰上。 注意:如果吸入溴化乙锭可能是致畸和毒性。阅读溴化乙锭安全技术说明书。 </li></ol></li><li>电泳<ol><li>加入200毫升的10倍MOPS缓冲至1800毫升的DDH 2 O，使一个1X MOPS运行缓冲液。 </li><li>使用1×MOPS运行缓冲液冲洗凝胶的孔中。 </li><li>预RUN T给他凝胶在20 V 5分钟。 </li><li>加载RNA样品到凝胶的孔中。 </li><li>用铝箔纸覆盖，避免光线照射。在35 V大约17小时运行（过夜） </li><li>检查和UV光下拍摄的凝胶。 </li></ol></li><li>转让<ol><li>切胶删除未使用的部分。 </li><li>制备500毫升的10×SSC缓冲液（250毫升20×SSC缓冲液和250mL DEPC处理的水）。 </li><li>用10倍的SSC缓冲液中以50rpm摇动洗凝胶两次20分钟。 </li><li>切1滤纸片大到足以用作灯芯纸，它从传送盘（ 图1）吸收转移缓冲液。 </li><li>切4张该滤纸的尺寸相同的凝胶。 </li><li>切成1片带正电荷的尼龙膜，以相同的尺寸的凝胶。 </li><li>标记在凝胶上的凹口和膜作为标记，以确保正确的方向。 </li><li>在2×SSC缓冲液浸泡的膜15分钟。 </li><li>倒入500毫升邻˚F10X SSC缓冲到传输托盘。 </li><li>铺设横跨玻璃板的大滤纸片并用转移缓冲液浸湿的滤纸。 </li><li>推出用吸管任何气泡。 </li><li>浸泡2片预先切割的滤纸在转移缓冲液，并将其放置在滤纸上的从步骤2.4.5的中心。删除任何气泡。 </li><li>莱凝胶顶面朝下放在滤纸。 </li><li>躺在凝胶的顶端膜上。对齐凝胶和膜的槽口。 </li><li>放置在膜的顶部2个预切割滤纸和湿用转移缓冲液的滤纸上。 </li><li>取出层之间的任何气泡。 </li><li>应用保鲜膜凝胶周围，以确保毛巾只吸收缓冲器通过凝胶和膜。 </li><li>堆叠在滤纸的顶部的纸毛巾。 </li><li>将一个超大的玻璃板上的毛巾上。 </li><li>放置一个重量上堆叠的顶部。 </li><li>保持过夜以允许所述RNA从凝胶转移到膜上。 </li></ol></li></ol> 3. N 6 -methyladenosine检测<ol><li>交联膜<ol><li>保持该膜在2×SSC缓冲液潮湿。 </li><li>将浸有10X SSC缓冲到UV交联剂滤纸2张。 </li><li>放置在滤纸上的顶部的膜，与所述RNA吸附面朝上。 </li><li>选择"autocrosslink模式"（120,000μJ），并按下"开始"按钮启动照射。 </li><li>检查在膜和凝胶用UV凝胶图像捕获器，以确认从该凝胶中的RNA转移到膜上。 </li></ol></li><li>免疫印迹<ol><li> 1小时阻断与吐温20（TBST）缓冲的Tris缓冲盐水在室温下，在5％非脂肪牛奶的膜。 </li><li>用TBST缓冲液三次洗15分钟。 </li><li>孵育膜M 6 A（隔夜&lt;青霉&gt; N 6 -methyladenosine）抗体溶液（1:4℃在5 1,000％非脂肪牛奶的TBST缓冲液）。 </li><li>用TBST缓冲液三次洗膜15分钟。 </li><li>孵育在HRP标记的驴抗兔抗体溶液（1:2,000，在5％非脂肪牛奶的TBST缓冲液）的膜1小时在室温下。 </li><li>用TBST缓冲液三次洗膜15分钟。 </li><li>应用增强的化学发光底物（每膜厘米2 0.125毫升）。 </li><li>捕捉与数字成像器的最佳设置的化学发光，根据制造商的说明26。 </li></ol></li><li>米6的量化<ol><li>测量使用ImageJ的软件相对米6化学发光强度。 <ol><li>根据ImageJ的软件菜单中，选择"文件"选项打开相关文件。 </li><li>从ImageJ的"矩形"工具和周围画一个框的信号。 </li><li>如果核糖体RNA不是实验的重点，避免用于计算的18S和28S核糖体RNA米6 A频带。 </li><li>点击"命令"和"1"来确认所选择的车道。 </li><li>按"命令"和"3"以显示所选的情节。 </li><li>点击"直线"工具，画线分割区分开。 </li><li>点击"棒"工具来记录测量。 </li><li>导出数据。 </li><li>规范化M 6 A的水平与来自溴化乙锭染色的18S核糖体RNA带。 </li></ol></li></ol></li></ol>

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Y., 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=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. 6, 18874 (2016).</li><li>Li, X., 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=Transcriptome-wide+mapping+reveals+reversible+and+dynamic+N-methyladenosine+methylome.">Transcriptome-wide mapping reveals reversible and dynamic N-methyladenosine methylome.</a> Nat Chem Biol. , (2016).</li><li>Sanschagrin, S., Yergeau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+of+16S+ribosomal+RNA+gene+amplicons.">Next-generation sequencing of 16S ribosomal RNA gene amplicons.</a> J Vis Exp. (90), (2014).</li><li>Kukutla, P., Steritz, M., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+ribosomal+RNA+for+mosquito+gut+metagenomic+RNA-seq.">Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.</a> J Vis Exp. (74), (2013).</li><li>Mishima, E., 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=Immuno-Northern+Blotting:+Detection+of+RNA+Modifications+by+Using+Antibodies+against+Modified+Nucleosides.">Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.</a> PLoS One. 10 (11), 0143756 (2015).</li><li>Klungland, A., Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+RNA+modifications+in+disease.">Dynamic RNA modifications in disease.</a> Curr Opin Genet Dev. 26, 47-52 (2014).</li></ol>

作者什么都没有透露。

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded8. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications27.
It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.
Transcriptome-wide mapping with m6A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA22. It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m6A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult18. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m6A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing30. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene's information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups. 
Modification of RNA is an important step in regulating transcriptional control31. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.
The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.
As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name "northeastern" blotting for the current technique.

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N6-methyladenosine (m6A) modifications were found to be abundant in eukaryotic transcriptomes1. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA2. The m6A modifications of mRNA influence their splicing3, translation4, degradation5, and localization2. Moreover, they affect ribosome biogenesis and microRNA function6. The evolutionary conservation of m6A modifications of RNA is noted in unicellular bacteria to multi-cellular humans7. Delineation of the roles of m6A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA8 also play critical roles in RNA metabolism. 
Circadian rhythms9, stem cell pluripotency10, triglyceride metabolism4, fibrosis11, obesity12, and major depression13 are a few examples of processes where m6A modifications are known to control outcomes. Many circadian clock gene transcripts have m6A sites14. Modulation of m6A methylase or demethylase elicits circadian period changes15. Mettl3, an m6A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage10. A deficiency of fat mass- and obesity-associated (FTO), an m6A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl44. These studies reveal that m6A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m6A modifications holds implications for therapeutic considerations in the future.
Several methods are available to measure m6A modifications of RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. Mass spectrometry is a sensitive tool for the detection of m6A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry21. Methyl-RNA immunoprecipitation and sequencing (m6A-Seq)22 immunoprecipitates fragmented RNAs with m6A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m6A landscapes. High-resolution mapping of m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m6A modifications at a single-nucleotide resolution23. Both methods provide details of m6A modification across the whole transcriptome, with specific genes' information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m6A-Seq alter the original RNA structure, which may affect native m6A levels. To detect global m6A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis18. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m6A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

<table><tbody><tr><td>RNaseZap solution</td><td>Ambion</td><td>AM9782</td><td>Protocol 1.1</td></tr><tr><td>TRI Reagent solution</td><td>Ambion</td><td>AM9738</td><td>Protocol 1.2.1.1</td></tr><tr><td>1-Bromo-3-chloropropane (BCP)</td><td>Sigma</td><td>B9673</td><td>Protocol 1.2.1.3</td></tr><tr><td>Ethanol</td><td>JTbaker</td><td>8006</td><td>Protocol 1.2.1.3</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>Protocol 1.2.1.3</td></tr><tr><td>GenElute mRNA Miniprep Kit</td><td>Sigma</td><td>MRN70</td><td>Protocol 1.2.2.1</td></tr><tr><td>Glycogen</td><td>Ambion</td><td>AM9510</td><td>Protocol 1.2.2.2</td></tr><tr><td>Sodium acetate</td><td>Fluka</td><td>71183</td><td>Protocol 1.2.2.2</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9930</td><td>Protocol 1.2.2.6</td></tr><tr><td>DEPC</td><td>Sigma</td><td>D5758</td><td>Protocol 2.1.1</td></tr><tr><td>Agarose</td><td>JT Baker</td><td>A426</td><td>Protocol 2.1.2</td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Protocol 2.1.3</td></tr><tr><td>37% formaldehyde Solution</td><td>Sigma</td><td>F8775</td><td>Protocol 2.1.3</td></tr><tr><td>EDTA, Disodium Salt</td><td>JT Baker</td><td>8993</td><td>Protocol 2.1.3 10x MOPS buffer</td></tr><tr><td>formamide</td><td>Sigma</td><td>F7503</td><td>Protocol 2.2.1</td></tr><tr><td>RNA Millennium Marker</td><td>Ambion</td><td>AM7150</td><td>Protocol 2.2.2</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>X328</td><td>Protocol 2.2.5</td></tr><tr><td>Ficoll PM400</td><td>GE Healthcare</td><td>17-0300-10</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>114391</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Xylene Cyanol FF</td><td>Sigma</td><td>X4126</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Sodium chloride dihydrate</td><td>JT Baker</td><td>3624</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Hybond Blotting Paper</td><td>GE Healthcare</td><td>RPN6101M</td><td>Protocol 2.4.4</td></tr><tr><td>GE Hybond-N+ membrane</td><td>GE Healthcare</td><td>RPN303B</td><td>Protocol 2.4.6</td></tr><tr><td>Stratalinker UV Crosslinker 2400</td><td>Stratagene</td><td>400075</td><td>Protocol 3.1.2</td></tr><tr><td>Gel Catcher 1500</td><td>ANT Technology</td><td>Gel Catcher 1500</td><td>Protocol 3.1.5</td></tr><tr><td>Anti-m&lt;sup&gt;6&lt;/sup&gt;A (N&lt;sup&gt;6&lt;/sup&gt;-methyladenosine)</td><td>Synaptic Systems</td><td>202003</td><td>Protocol 3.2.3</td></tr><tr><td>Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td><td>GE Healthcare</td><td>NA934</td><td>Protocol 3.2.5</td></tr><tr><td>ChemiDoc MP System</td><td>BIO-RAD</td><td>1708280</td><td>Protocol 3.2.8</td></tr></tbody></table>

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.

后在正常光暗昼夜阶段14天，野生型小鼠被放置在不断的黑暗。从肝脏中的RNA在4小时的时间间隔进行采样，并与修改的Northern印迹分析。个rRNA，mRNA的和小RNA的甲基化被清楚地检测（ 图2）。不同的24小时时间之间（CT）的比较可以与18S rRNA的标准被精确地计算。有在rRNA基因的mRNA和小RNA米6 A水平的稳健昼夜振荡。 以避免的rRNA递上的干扰，多聚腺苷酸化的RNA可以被纯化，如在步骤1.2.2。纯化后，rRNA基因可以大大消除，以允许其它RNA（ 图3）的更好的可视化。 <img alt="图1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt;的图1:装配印迹传送装置。用于印迹的RNA与膜中的毛细管转移单元被示出。 <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54672/54672fig2.jpg" /> 图2:在C57BL / 6J小鼠的M 6 A水平的昼夜节律。该图示出了第m 6从野生型小鼠的肝脏总RNA的印迹上的暗暗相位的第一天处死后的正常光黑暗阶段的14天内，在4小时的间隔。量化是使用M 6 18S和28S rRNA基因之间的图像密度差完成。 m个6的丰度，从底部的甲醛凝胶归到18S rRNA条带。等="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54672/54672fig3.jpg" /> 图3:M 6一个印迹带或不带rRNA基因。代表m总RNA和从野生型小鼠的肝脏中聚腺苷酸化RNA的6的印迹被示出。 <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1 </td><td colspan="3"> 10X MOPS缓冲液（pH 7.0，避光） </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> MOPS </td><td> 41.85 </td><td> G </td><td> 0.2M的</td></tr><tr><td></td><td colspan="3">醋酸钠</td><td> 4.1 </td><td> G </td><td>0.05M的</td></tr><tr><td></td><td colspan="3"> EDTA，二钠盐</td><td> 3.7 </td><td> G </td><td> 0.01M的</td></tr><tr><td></td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">总</td><td> 1 </td><td>大号</td><td></td></tr><tr><td></td><td colspan="3">在室温下搅拌</td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20×SSC缓冲液（pH7.0）中</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">氯化钠</td><td> 175.3 </td><td> G </td><td> 3米</td></tr><tr><td></td><td colspan="3">柠檬酸钠</td><td> 88.3 </td><td> G </td><td> 0.3M的</td></tr><tr><td></td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">总</td><td> 1 </td><td>大号</td><td></td&gt; </tr><tr><td></td><td colspan="3">在室温下搅拌，然后高压灭菌</td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3">示踪染料</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10X MOPS缓冲液</td><td> 500 </td><td> μL </td><td> 1X </td></tr><tr><td></td><td colspan="3">聚蔗糖400 </td><td> 0.75 </td><td> G </td><td> 15％ </td></tr><tr><td></td><td colspan="3">溴酚蓝</td><td> 0.01 </td><td> G </td><td> 0.2％ </td></tr><tr><td></td><td colspan="3">二甲苯青</td><td> 0.01 </td><td> G </td><td> 0.2％ </td></tr><tr><td></td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">总</td><td>五</td><td>毫升</td><td></td></tr><tr><td></td><td colspan="3">储存在-20°C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">在1比例稀释:DEPC 1000在DDH 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">在室温下搅拌过夜，然后高压灭菌</td><td></td><td></td><td></td></tr><tr><td>五</td><td colspan="3">样品缓冲液（避光） </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10X MOPS缓冲液</td><td> 200 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> 37％的甲醛</td><td> 270 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3">甲酰胺</td><td> 660 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3">储存在-20°C </td><td></td><td></td><td></td></tr></tboDY&gt; </table> 表1:缓冲器和解决方案。

Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

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

中描述了RNA测定N 6 -methyladenosine（M 6 A）修改的改进RNA印迹法。当前的方法可以检测在各种实验设计在不同的RNA修改和控制。

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 ...

a-method-for-measuring-rna-n6-methyladenosine-modifications-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

10.8K 次观看.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine. 中描述了RNA测定N 6 -methyladenosine（M 6 A）修改的改进RNA印迹法。当前的方法可以检测在各种实验设计在不同的RNA修改和控制。 

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

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 ...

科研

生物化学

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 ...

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 ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

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 ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

用核磁共振波谱探索精氨酸甲基组

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

癌症研究

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

使用 HPLC-MS/MS 进行核苷/核苷酸含量测量的工厂样品制备

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

rRNA 2'-O-甲基化的脱氧核糖核酸依赖性分析

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

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 ...

一种测量方法RNA

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体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

RNA甲基转移酶活性分析的无抗体测定

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

甲基化RNA免疫沉淀分析研究m^5C在阿拉伯的修改

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

用核磁共振波谱探索精氨酸甲基组

使用 HPLC-MS/MS 进行核苷/核苷酸含量测量的工厂样品制备

rRNA 2'-O-甲基化的脱氧核糖核酸依赖性分析

使用质谱法表征单个神经元中的RNA修饰