Name: antibody-free assay for rna methyltransferase activity analysis
Uploaded: 2019-07-09T15:00:00
Description: Watch this Scientific Journal Video about Antibody-Free Assay for RNA Methyltransferase Activity Analysis at JoVE.com

作者要感谢图里亚·坎蒂·德布纳特博士对ChemDraw的帮助。Xhemalée 实验室的研究得到国防部 - 国会指导医学研究计划 - 乳腺癌突破奖 (W81XWH-16-1-0352), NIH 格兰特 R01 GM127802 和细胞和研究所的启动资金支持美国奥斯汀得克萨斯大学分子生物学和自然科学学院。

1. 靶着RNA的体外转录和凝胶纯化
<ol><li>使用既定的分子克隆技术12或试剂盒,将感兴趣的序列克隆成含有T7和/或SP6启动子的质粒。</li>
	<li>为体外转录的DNA模板线性化
	<ol>
<li>通过质粒的PCR放大感兴趣的序列,使用引物,通过插入物包括T7启动子区域,如前面描述的20-30 bp上游和下游。</li>
		<li>或者,根据RE供应商提供的说明,使用限制酶(RE)切割部位在刀片?...

<ol>
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D., 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=5'+UTR+m(6)A+Promotes+Cap-Independent+Translation.">5' UTR m(6)A Promotes Cap-Independent Translation.</a> Cell. 163 (4), 999-1010 (2015).</li><li>Wang, 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=N(6)-methyladenosine+Modulates+Messenger+RNA+Translation+Efficiency.">N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency.</a> Cell. 161 (6), 1388-1399 (2015).</li><li>Alarcon, C. R., Lee, H., Goodarzi, H., Halberg, N., Tavazoie, S. 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在这里,报告了一种简单而可靠的方法,用于对特定转录本进行RNA甲基转移酶活性的体外验证。该测定利用了S-腺苷可对甲基组供体进行三聚酯(图1)的事实,使得甲基化RNA在不使用抗体的情况下能够准确检测。然而,需要注意的是,这种测定不能指示酶甲基化的残留物或化学组。为了识别或验证特定残留物是否甲基化,其他方法,如突变分析、逆转录块或RNA基质谱分析,可与RNA甲基转移...

脱氧核糖核酸、RNA和蛋白质受到严格调节基因表达1的修饰。在这些修饰中,甲基化发生在所有三种生物聚合物上。在过去的三十年里,DNA和蛋白质甲基化得到了很好的研究。相比之下,鉴于写、擦除或结合RNA甲基化的蛋白质在发育和疾病2中的重要作用,对RNA甲基化的兴趣直到最近才重新燃起。除了在丰富的核糖体和转移RNA中更知名的功能,RNA甲基化通路调节特定的信使RNA稳定性3,4,拼接5和翻译6,7,miRNA处理8,9和转录暂停和释放10,11。
这里报告了一种简单而可靠的方法,用于在分子生物学实验室的设置中对RNA甲基转移酶活性进行体外验证(如图1所示)。许多研究评估RNA甲基转移酶的活性,通过点斑点与抗体对RNA修饰的兴趣。然而,点泡在用RNA甲基转移酶孵育时不能验证RNA的完整性。这一点很重要,因为即使对核酸酶的重组蛋白进行轻微污染,也会导致部分RNA降解和混淆结果。此外,即使是高度特异性的RNA修饰抗体也能识别具有特定序列或结构的未修饰RNA。这里报道的体外RNA甲基转移酶测定利用了S-腺苷蛋氨酸可以在甲基组供体上三聚酯(图1)的事实,使得甲基化RNA在不使用抗体的情况下能够准确检测出来.提供在体外转录和纯化兴趣转录和测试该转录本的甲基化由感兴趣的酶。该方法灵活可靠,可根据任何给定项目的需求进行调整。例如,在体外转录和纯化RNA,化学合成RNA,也可以使用细胞RNA。此测定以闪烁计数的形式提供定量信息,并通过显示甲基化RNA在凝胶上的确切运行位置提供定性信息。这可以为RNA甲基转移酶的功能提供独特的见解,特别是在使用细胞RNA作为基质时,因为它提供了一种直接观察甲基化目标RNA或RNA的大小的方法。

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			<td height="21" style="height:21px;width:216px;">10 bp DNA Ladder</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">10821-015</td>
			<td style="width:353px;">10 bp DNA Ladder kit.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">10% Ammonium Persulfate (APS)&#160;</td>
			<td style="width:121px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td style="width:353px;">For urea denaturing polyacrylamide gel (For 10 mL, dissolve 1g in 8 mL of milliQ water; adjust volume to 10 mL with milliQ water; filter the solution using a 10 mL syringe equiped with a 0.45 &#181;m filter).</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">10X TBE Buffer</td>
			<td style="width:121px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td style="width:353px;">For urea denaturing polyacrylamide gel (For 1L, add 108 g of Tris Base, 55 g of Boric Acid to a cylinder with a stir bar; add 800 mL of distilled water and let dissolve; add 40mL of 0.5 M Na2EDTA (pH 8.0); adjust volume to 1L with milliQ water; filter the solution using a 0.22&#181;m filter).</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">10X TBS</td>
			<td style="width:121px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td style="width:353px;">For 1L, add 60.5 g of Tris Base, 87.6 g of NaCl to a cylinder with a stir bar; add 800 mL of distilled water and let dissolve; adjust pH to 7.5 with concentrated HCl; adjust volume to 1L with milliQ water; filter the solution using a 0.22 &#181;m filter.&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Acrylamide: Bis-Acrylamide 29:1 (40% Solution/Electrophoresis), Fisher BioReagents</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:161px;">BP1408-1</td>
			<td>For urea denaturing polyacrylamide gel.</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">ADENOSYL-L-METHIONINE, S-[METHYL-3H]; (SAM[3H])</td>
			<td style="width:121px;">Perkin Elmer</td>
			<td style="width:161px;">NET155V250UC</td>
			<td style="width:353px;">For in vitro methylation of RNA; Concentration = 1.0 mCi/mL; Specific activity = 17.1 Ci/mmol; Molarity= 
			(1.0 Ci/L)/(83.2 Ci/mmol) = 0.0584 mmol/L = 58.4 &#181;M.&#160;&#160; Upon receipt of the frozen 3H-SAM tube, thaw it at 4&#176;C, make 20 &#181;L aliquots, and freeze them at -30&#176;C. Never refreeze and reuse a partially used aliquot.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amersham Hypercassette Autoradiography Cassettes</td>
			<td style="width:121px;">GE Healthcare</td>
			<td style="width:161px;">RPN11649</td>
			<td style="width:353px;">For autoradiogram gel exposure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amersham Hyperfilm MP</td>
			<td style="width:121px;">GE Healthcare</td>
			<td style="width:161px;">28906846</td>
			<td style="width:353px;">For autoradiogram gel exposure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beckman Scintillation Counter</td>
			<td>Beckman</td>
			<td>LS6500</td>
			<td style="width:353px;">For liquid scintillation count.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biorad Mini Horizontal Electrophoresis System</td>
			<td>Biorad</td>
			<td>1704466</td>
			<td style="width:353px;">Mini Horizontal Electrophoresis System.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">cOmplete Mini EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td style="width:121px;">Roche Applied Science</td>
			<td style="width:161px;">4693159001</td>
			<td style="width:353px;">For a 20X solution, dissolve 1 tablet in 0.525 mL of nuclease free water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Criterion Cell</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:161px;">345-9902</td>
			<td>RNase free empty cassette for polyacrylamide gel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Criterion empty Cassettes</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:161px;">1656001</td>
			<td>Vertical midi-format electrophoresis cell.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DeNovix DS-11 Microvolume Spectrophotometer</td>
			<td style="width:121px;">DeNovix</td>
			<td style="width:161px;">DS-11-S</td>
			<td style="width:353px;">Microvolume Spectrophotometer for measuring DNA and RNA concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ecoscint Original</td>
			<td>National Diagnostics</td>
			<td style="width:161px;">LS-271</td>
			<td style="width:353px;">For liquid scintillation count.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand 7mL HDPE Scintillation Vials</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:161px;">03-337-1</td>
			<td style="width:353px;">For liquid scintillation count.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluoro-Hance-Quick Acting Autoradiography Enhancer</td>
			<td style="width:121px;">RPI CORP</td>
			<td style="width:161px;">112600</td>
			<td style="width:353px;">For autoradiogram gel pretreatment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel dryer</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:161px;">1651745</td>
			<td style="width:353px;">For drying gel.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Gel Loading Buffer II</td>
			<td style="width:121px;">Ambion</td>
			<td style="width:161px;">AM8547</td>
			<td style="width:353px;">For loading RNA in denaturing polyacrylamide urea gel (composition: 95% Formamide, 18 mM EDTA, and 0.025% SDS, Xylene Cyanol, and Bromophenol Blue).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GeneCatcher disposable gel excision tips</td>
			<td style="width:121px;">Gel Company</td>
			<td style="width:161px;">NC9431993</td>
			<td>For removing bands from agarose and polyacrylamide gels.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Megascript Kit</td>
			<td style="width:121px;">Ambion</td>
			<td style="width:161px;">AM1333</td>
			<td>For in vitro transcription with T7 RNA polymerase.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Perfectwestern Extralarge Container</td>
			<td style="width:121px;">Genhunter Corporation</td>
			<td style="width:161px;">NC9226382 (clear)/ NC9965364 (black)</td>
			<td>Gel staining box.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pRZ</td>
			<td style="width:121px;">Addgene</td>
			<td style="width:161px;">#27663</td>
			<td style="width:353px;">Plasmid for producing in vitro transcripts with homogeneous ends</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen RNeasy MinElute Cleanup</td>
			<td style="width:121px;">Qiagen</td>
			<td style="width:161px;">74204</td>
			<td style="width:353px;">For RNA clean-up, use modified protocol provided in the protocol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAquick Gel Extraction Kit (50)</td>
			<td style="width:121px;">Qiagen</td>
			<td style="width:161px;">28704</td>
			<td style="width:353px;">Kit for gel extraction and clean up of dsDNA fragment used for in vitro transcription.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Saran Premium Plastic Wrap</td>
			<td style="width:121px;">Saran Wrap</td>
			<td style="width:161px;">Amazon</td>
			<td style="width:353px;">For drying gel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR Gold</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">S11494</td>
			<td>Ultra sensitive nucleic acid gel stain.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR Safe</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">S33102</td>
			<td>Nucleic acid gel stain.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TE</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">93283-100ML</td>
			<td>10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:161px;">110-18-9</td>
			<td style="width:353px;">For urea denaturing polyacrylamide gel.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Thermomixer with SMARTBLOCK 24X 1.5mL TUBES</td>
			<td>eppendorf</td>
			<td style="width:161px;">5382000023/5361000038</td>
			<td style="width:353px;">For temperature controlled incubation of 1.5 mL tubes.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TOPO TA Cloning Kit</td>
			<td style="width:121px;">life technologies</td>
			<td style="width:161px;"></td>
			<td style="width:353px;">Kits for fast cloning of Taq polymerase&#8211;amplified PCR products into vectors containing T7 and/or SP6 promoters for in vitro RNA transcription.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TURBO DNase (2 U&#8260;&#181;L)</td>
			<td style="width:121px;">Ambion</td>
			<td style="width:161px;">AM2238</td>
			<td>For DNA removal from in vitro transcription reactions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">51456-500G</td>
			<td>For urea denaturing polyacrylamide gel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Whatman 3MM paper</td>
			<td style="width:121px;">GE Healthcare</td>
			<td style="width:161px;">3030-154</td>
			<td style="width:353px;">Chromatography paper for drying gel.</td>
		</tr>
	</tbody>
</table>

antibody-free assay for rna methyltransferase activity analysis

RNA有100多个化学上不同的修饰,其中三分之二由甲基化组成。由于对RNA修饰,特别是甲基化的兴趣,由于在与疾病和癌症相关的生物过程中书写和清除酶所起的重要作用,重新出现了。这里提供了一种敏感的体外测定,用于准确分析RNA甲基化作家在合成或体外转录RNA上的活性。这种测定使用三聚体形式的S-腺苷-蛋氨酸,导致甲基化RNA直接贴上三聚糖标签。低能量的三聚能使该方法安全,而且预先存在的三聚物信号扩增方法,使得在不使用通常容易产生伪影的抗体的情况下,可以量化和可视化甲基化RNA。虽然这种方法是为RNA甲基化而编写的,但很少的调整使它适用于研究其他可放射性标记的RNA修饰,例如14 C乙酰辅酶A的RNA乙酰化。甲基化条件,抑制与小分子抑制剂,或RNA或酶突变体的作用,并提供一个强大的工具,以验证和扩大在细胞中取得的结果。

体外转录反应 图 2A表示与 7SK snRNA 的 T7 RNA 聚合酶的体外转录反应的典型运行,该酶是相对较短(331 nt)和结构高度结构化的 RNA。如该原始图像所示,有多个不需要的波段,时间都短于 7SK,可能是随机转录启动或终止事件造成的。因此,体外转录反应后的凝胶纯化对于获得干净的RNA样本非常重要,如图2B所示。 此时,感兴趣...

Watch this Scientific Journal Video about Antibody-Free Assay for RNA Methyltransferase Activity Analysis at JoVE.com

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

这里描述了一种无抗体的体外测定,用于直接分析合成或体外转录RNA上的甲基转移酶活性。

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 ...

RNA有100多个不同的修饰，其中三分之二是甲基化。该协议为对RNA甲基化编写器活性进行灵敏和准确的分析提供了一种方法。这种易于使用的技术使我们能够量化和可视化甲基化RNA，而无需使用抗体，这些抗体通常容易成为伪影。展示这个程序的将是萨曼莎·谢尔顿，一个来自我实验室的研究生。为了开始这个程序，在1.5毫升的冰上管中设置100微升RNA甲基转移酶测定，如文本协议中所述。将管子漩涡彻底混合，在200倍g下离心5秒钟，收集管底的溶液。在37摄氏度下孵育管子两小时。然后，使用列纯化清洁反应，如文本协议中所述。要执行液体闪烁计数，请设置闪烁计数机架，每个样品一小瓶，背景测量一小瓶，刷卡测试一小瓶。用五毫升的闪烁计数溶液填充小瓶。将每个洗洗放射性RNA样品的10微升加入一个小瓶中。拧紧盖子，轻轻混合。要准备刷卡测试小瓶，请在手术过程中使用的所有表面和设备上彻底擦拭棉签。将这些棉签加入装满闪烁溶液的小瓶中，然后拧紧盖子。要在闪烁计数器上运行样品，请打开计数器罩，将机架插入机器，然后关闭发动机罩。选择计数单个机架。选择"选择用户程序"。选择或创建一个程序，测量钛60秒，并击中计数机架。重复闪烁计数三次。在此之后，准备并重新运行尿素变性聚丙烯酰胺凝胶，如文本协议中所述。将20微升放射性RNA材料转移到含有20微升2X凝胶加载缓冲液的1.5毫升管中。准备梯子后，在70摄氏度下孵育样品15分钟。在样品加载之前，立即将凝胶的孔再次清洗，将缓冲液从凝胶罐向下移液缓冲到凝胶的孔中。然后，将 20 微升的已准备好的梯子、20 微升的样品和 20 微升的 1X 凝胶加载缓冲液加载到凝胶的通道中。根据聚丙烯酰胺百分比，以 100 伏运行凝胶 60 至 180 分钟。凝胶运行完成后，从盒式磁带上取出凝胶，并将其放在一个装有 50 毫升 1X TBE 缓冲液的盒子里，并含有 5 微升超敏核酸凝胶污渍。在摇杆上孵育五分钟，以染色RNA。在此之后，小心地将凝胶从包装盒中拿出来，并放在凝胶成像系统的紫外线透光器上，井向上，梯子在左侧。将相机对焦在凝胶上，打开紫外线，然后拍摄凝胶的图像。然后，关闭 UV 曝光，然后将图像另存为 TIFF 文件。将凝胶放回包装盒中，取出 TBE，并在室温下将凝胶与 50 毫升固定溶液固定在摇杆上 30 分钟。接下来，轻轻地将凝胶移动到一个新鲜的黑盒，其中包含 25 毫升的自造增强溶液。在室温下在摇杆上孵育30分钟。轻轻提起凝胶，面朝下放在一块塑料包装上，井向上，梯子放在右侧。将两张色谱纸放在凝胶背面，然后翻转整个堆栈。将凝胶干燥机预热至 80 摄氏度。将凝胶干燥器上的塑料盖移回。将整个堆叠件插入塑料盖下，然后向下移动塑料盖以创建密封件。在80摄氏度下干燥凝胶一小时。然后，关闭凝胶干燥器，轻轻取出堆叠。取出包装纸和第二色谱纸。将剩余的色谱纸与干凝胶一起用自动放射图盒装上。在黑暗的房间里，添加一张自摄影胶片。将盒式磁带存放在负 80 摄氏度，并根据信号强度在适当时间开发胶片。一旦薄膜被开发，把它放在盒式磁带的顶部，并使用实验室标记仔细标记凝胶的四个边缘，每个井，以及XC和BB染料的位置。以每英寸 300 或 600 像素的分辨率扫描胶片，然后将图像另存为 TIFF 文件。在这项研究中，证明一种无抗体的测定可以分析RNA甲基转移酶的活性。与7SK小核RNA的T7RNA聚合酶进行体外转录反应的典型运行显示相对短且结构高的RNA。有多个不需要的波段，短和长于7SK，可能是由随机转录启动或终止事件引起的。因此，体外转录反应后凝胶纯化对于获得干净的RNA样品非常重要。此时，感兴趣的RNA可以通过相对于梯子的位置来识别，并通过凝胶纯化进行纯化。此处显示了使用推荐RNA和蛋白质浓度的下限的代表性RNA甲基转移酶测定。此测定允许从闪烁计数的定量结果，以及从自动放射图的定性结果。在这里，一种以甲基酸盐7SK已知的RNA甲基转移酶被证明也能够使甲基六甲酸酯U6。此外，正如最近对7SK所示，组蛋白H4与MPCE的结合也抑制U6甲基化。这可以在闪烁计数和自动放射图中观察到，显示此协议的鲁棒性。RNA 容易降解，因此请确保使用无 RNase 试剂并彻底清洁所有表面和设备。例如，从这种甲基转移酶测定中获得的放射性标记RNA可以直接用于评估RNA脱乙基酶活性或RNA结合活性。这种方法允许研究人员测试不同因素对RNA甲基转移酶活性的影响，包括点突变和小分子抑制剂治疗。此方法使用放射性物质，因此请务必佩戴适当的 PPE，在处理和处理放射性物质时要小心。

Research

JoVE Journal

Genetics

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

科研

遗传学

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

的转录分析<em&gt;ç</em&gt;。<em&gt;线虫</em&gt; RNA测序数据通过炫套房的银河项目

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

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

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 ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

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

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

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 ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

酿酒酵母4-硫尿嘧啶代谢标记及新合成 mRNA 作为 RNA 聚合酶 II. 活性的一种代理

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

CRISPR Guide RNA Cloning for Mammalian Systems

哺乳动物系统 CRISPR 指南 RNA 克隆

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

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 ...