Name: investigation of rna synthesis using 5-bromouridine labelling and immunoprecipitation
Uploaded: 2018-05-03T13:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation at JoVE.com

Lundbeck 基金会、奥胡斯大学、Dandrite 和 Lundbeck 支持这项工作。

注: 在室温下执行所有步骤, 除非另有说明, 并在冰上或冰箱中保持缓冲 (除含有 SDS 的洗脱缓冲液)。该协议分为5个部分: RNA 样品的制备;制备用于 IP 的珠子;老兄标记 RNA 与珠子的结合;3步苯酚-苯酚/氯仿-氯仿萃取分离纯化老兄标记 RNARNA 分析 (在这个例子中使用 RT qPCR)
1. RNA 样品的制备
<ol><li>使用自动细胞计数器和种子350万细胞计数 HEK293T 细胞在 10 cm 培养皿10毫升生长培养基 (Dulbecco 的改良鹰的培养基补充10% 胎小牛血清和50µg/毫升青霉素/链霉素), 以获得总 > 40 µg RNA48小时后提取。保持单元格在37°c 和 5% CO2。</li>
	<li>48小时后播种, 吸8毫升生长培养基, 并留2毫升后, 以避免干燥的细胞。添加2毫米老兄和任何治疗8毫升吸气生长培养基, 并从细胞中取出剩余2毫升, 然后再应用含有生长介质的8毫升老兄。避免使用新鲜的媒介在应用老兄对细胞, 因为这可能导致基因表达的变化。</li>
	<li>用老兄和治疗方法孵化细胞1小时。用2毫升0.05% 胰蛋白酶将5毫升的胰蛋白酶处理中的细胞洗净。添加8毫升的生长培养基以停止反应, 将细胞转移到15毫升管, 并在 1000 x g 上将细胞向下旋转2分钟。</li>
	<li>使用 RNA 隔离套件 (请参阅材料表) 或任何其他首选方法, 从细胞颗粒中取出上清并提取总 RNA, 并在无 RNase 的 H2中洗脱将 RNA 存储在-80 摄氏度, 直到准备好进行。</li>
</ol>2. 制备用于 IP 的珠子
<ol><li>在吹打和旋流混合过程中, 为要调查的样品数加上一个额外的损失, 准备批珠。并用重悬抗鼠 IgG 磁性珠彻底的旋流搅拌和取出20µL 珠每样品在一个 RNase 免费1.5 毫升管。放置1.5 毫升管在一个磁性立场和离开大约二十年代为珠子收集在边。</li>
	<li>取出上清液, 从磁支架取出管, 在400µL 1x 老兄 IP 缓冲器中并用重悬吹打。在台式离心机中非常简单地旋转管子2秒, 将其放回磁性支架中, 取出上清。重复洗涤步骤一次。</li>
	<li>用肝素阻止不特异的珠子绑定。并用重悬珠在1毫升1x 老兄-IP + 1 毫克/毫升肝素缓冲, 并离开旋转30分钟. 洗涤珠一次, 如在步骤2.2。 注: 肝素是一种负电荷聚合物, 它将与 rna 竞争以进行装订。如果下游应用程序没有排序, 也可以使用 tRNA 或其他不相关的 rna。</li>
	<li>并用重悬1毫升1x 老兄 IP 缓冲器中的珠子, 并添加1.25 µg/样本抗α Bromodeoxyuridine 抗体, 这也承认老兄。在旋转时孵化1小时的珠子, 或在室温下过夜。</li>
	<li>在步骤2.2 中洗三次珠子。并用重悬在50µL/样品1x 老兄-IP 缓冲器补充1毫米老兄和离开旋转30分钟, 以降低抗体的灵敏度与珠, 从而在 IP 期间不特异绑定的风险。</li>
	<li>将珠子洗净三倍于步骤 2.2, 并用重悬50µL/样本 1 xbru IP 缓冲器。</li>
</ol>3. 老兄标记 RNA 对珠子的约束力
<ol><li>用紫外可见分光光度法测量步骤1.3 中 rna 提取物中的 rna 浓度, 并将 RNase 中的每个样品中的40µg rna 稀释为200µL 的总体积.</li>
	<li>热 rna 样品2分钟在80°c 到变性 RNA 并且简要地旋转在桌上离心机。添加200µL 2x 老兄-IP + BSA/RNAse 抑制剂。</li>
	<li>添加50µL 悬浮和准备珠从步骤2.6 到每个样品和离开旋转 1 h. 洗涤珠四倍, 在步骤2.2。</li>
</ol>4. RNA 的洗脱和纯化3步苯酚-苯酚/氯仿-氯仿萃取
<ol><li>
洗脱 RNA 和执行苯酚提取
	<ol>
<li>并用重悬在200µL 洗脱缓冲液中的珠子洗脱 RNA, 然后迅速添加200µL 苯酚, pH 6.6 (警告: 毒性, 见步骤 4.1.2) 删除任何非 RNA 分子从样本。</li>
		<li>旋转混合样品, 并旋转3分钟在 2.5万 x g 在一个台式离心机。 注: 苯酚是有毒的, 应处理在通风罩上, 佩戴皮肤保护。稍微酸性的 pH 值可以确保提取的只是 RNA, 而不是 DNA。由于电荷的亲水性性质, RNA 将留在上水相中。蛋白质的疏水性核心会结合苯酚, 并移动到较低的有机酚相。</li>
	</ol>
</li>
	<li>
执行苯酚-氯仿萃取
	<ol>
<li>吸入尽可能多的上部阶段尽可能 (大约190µL 200 µL, 根据经验在处理) 和移动它到一个管子与200µL 苯酚或氯仿, pH 6.6 (警告: 毒物, 看见注意)。请确保在样品中吸出相同的量。</li>
		<li>旋转混合样品和旋转3分钟在 2.5万 x g。 注: 氯仿是有毒的, 应处理在通风罩上, 佩戴皮肤保护。氯仿是添加到去除苯酚, 这可能影响到下游分析 RNA 通过抑制反向转录14和聚合酶链反应15。</li>
	</ol>
</li>
	<li>执行氯仿萃取通过吸水上阶段之前 (现在约180µL), 并转移到一个管含有200µL 氯仿。旋转混合样品和旋转3分钟在 2.5万 x g。</li>
	<li>吸入水上相之前 (现在约170µL) 和转移到一个管, 其中包含17µL (1/10 卷) 3 米 CH3COONa, pH 6.0。从水溶液中中和并沉淀 RNA, 加入2µL 糖原 (20 毫克/毫升), 与 RNA 沉淀在一起, 使 rna 颗粒更加可见。</li>
	<li>添加500µL 96% 乙醇, 以增加盐离子和 RNA 之间的结合, 并通过反转管几次。在干冰上将管子在-20 摄氏度或15分钟内过夜。</li>
	<li>旋转样品在 2.5万 x g, 4 °c 为30分钟丢弃上清, 不干扰颗粒和洗涤颗粒在185µL 75% 乙醇去除任何残余盐。旋转 2.5万 x g, 4 °c 5 分钟。</li>
	<li>完全放弃上清, 不干扰球团, 使颗粒干燥5-10 分钟, 用开盖。并用重悬在10µL 无 RNaseH2 O 应用在管的一侧, 以避免干扰颗粒。</li>
</ol>5. RNA 分析
<ol><li>利用 cdna 反向转录试剂盒 (参见材料表) 或任何其他首选方法, 使用2µL rna 样本和1µg 酵母总 rna 作为载体 rna 进行 cdna 合成反应 (如果用 cdna 进行下一代测序), 则使用该基因制备 cdna。使用1µg rna 没有酵母 rna 为对应的输入的 cDNA 准备。</li>
	<li>稀释 cdna 1:25 和混合9µL 稀释 cdna 与10µL qPCR 主混合和1µL 荧光探针, 两者都在材料表中指定。 注: cdna 稀释依赖于 cdna 的制备和 qPCR 方法的应用。</li>
	<li>运行以下 qPCR 程序 (例如,在7500快速实时 PCR 系统或等效项): 2 分钟在50°c, 二十年代在95°c, 40 个周期 3 s 在95°c 和三十年代在60°c。</li>
</ol>

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作者没有什么可透露的。

该协议描述了使用老兄-IP 来确定 rna 生产通过标签新产生的 rna 1 小时与老兄, 然后立即 RNA 提取和免疫沉淀老兄标记 rna。此方法以前在引用16中进行了描述, 本文还包括一些附加步骤以提高 IP 特异性和 RNA 纯度, 方法是先处理低浓度老兄的珠子以及苯酚-氯仿净化步骤, 以增加 RNA 纯度跟随 IP。此外, 我们还通过比较老兄和非老兄治疗细胞中捕获的 RNA, 来展示老兄的特异性。苯酚-氯?...

5-Bromouridine (老兄) 免疫沉淀 (IP) 允许在简短的标签期间, 对细胞生理学没有或非常有限影响的细胞中 RNA 的产生进行研究 1, 2.该方法的基础是将合成苷衍生物老兄纳入新合成的 rna, 然后使用抗老兄抗体的标记 RNA 的 IP (图 1)。
几十年来, 人们已经知道蛋白质合成可以被转录调控, 转录因子的存在在50年前被推测为3。今天, 已知一些疾病是由转录和 rna 稳定性的失调引起的 (补充图 S1 在参考4), 并且测量 RNA 生产的变化的能力在了解疾病是非常重要的发展。
另一方面, 调控 RNA 生产的工具为治疗因过少或过多的蛋白质表达或诸如帕金森氏病 (PD) 等蛋白质积累而引起的疾病提供了新的可能性。由于α-synuclein 基因的增殖导致家族 pd5, synuclein 中的主要蛋白质积累为不溶性聚集体, α-synuclein 的水平与该病直接相关。此外, α synuclein 聚集在浓度依赖性的方式。下调的α synuclein mRNA 水平是一个有趣的治疗策略, 已经成功地获得了利用 RNA 干扰, 以减少神经元细胞损失的 PD 啮齿动物模型6,7。
重要的是要能够测量 RNA 生产的变化引起的任何疾病或治疗干预。测量 RNA 稳态水平的艺术方法, 如反向转录和定量聚合酶链反应 (RT-qPCR), 不能区分转录调节引起的变化或改变RNA 的稳定性。一种广泛应用的 RNA 衰变率调查方法是使用诸如α amanitin 或放线菌 D 等化合物的转录机械阻断, 然后再测量衰变 rna。然而, 在细胞中转录的整体堵塞, 如诱导凋亡8,9, 也存在一些问题。除细胞毒作用之外, 转录抑制剂还存在几个技术问题, 例如 amanitin 细胞的缓慢摄取和放线菌 D 缺乏特异性 (参考文献10)。
为了避免转录的整体堵塞, 核苷酸类似物已经被使用, 如 5-乙炔苷 (5-欧盟), 4-thiouridine (4) 和老兄。这些都很容易被哺乳动物细胞吸收并纳入新合成的 rna 中, 从而在特定的时间范围内对 rna 进行脉冲标记。老兄的毒性低于 5-欧盟和 4, 使其成为首选的模拟选择11,12。
老兄可以用来研究 rna 合成的速率和稳定性, 从而区分总 rna 变化的根本原因。本文将重点研究 rna 合成的测量, 并参考2 , 以了解 rna 稳定性的研究情况。为了研究 RNA 的合成, 细胞用老兄例如1 小时的简单标记, 后跟老兄-IP1 (图 1C)。这使得在短标签时间内合成的 rna 的测量和观察到的变化将比通过 qPCR 测量总 rna 的变化来更好地估计 rna 合成中的规则。然而, 应该提到的是, 即使标签时间, 例如, 只有1小时, 退化仍然可能对观察到的 RNA 水平产生影响。
老兄-IP 实验的 RNA 适用于 RT qPCR1或下一代测序2,13的下游分析。其他标准 rna 检测方法, 如北印迹或核糖核酸保护试验, 也可能适用于某些 rna。

<table><tbody><tr><td>Ribolock Rnase inhibitor</td><td>ThermoFisher</td><td>EO0381</td><td></td></tr><tr><td>Dynabeads M-280 Sheep anti-mouse IgG</td><td>Life Technologies</td><td>11202D</td><td></td></tr><tr><td>&amp;alpha;-Bromodeoxyuridine mouse antibody</td><td>BD Bioscience</td><td>555627</td><td></td></tr><tr><td>Nanodrop 1000</td><td>Thermo Fisher</td><td></td><td>Ultraviolet-visible spectrophotometry</td></tr><tr><td>Water-Saturated Phenol pH 6.6</td><td>ThermoFisher</td><td>AM9712</td><td></td></tr><tr><td>Phenol:Chloroform:IAA 25:24:1 pH 6.6</td><td>ThermoFisher</td><td>AM9732</td><td></td></tr><tr><td>High Pure RNA isolation Kit</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher</td><td>4368814</td><td></td></tr><tr><td>Taqman Fast Advanced Master Mix</td><td>ThermoFisher</td><td>4444557</td><td>qPCR Master Mix</td></tr><tr><td>Taqman RNA probes</td><td>ThermoFisher</td><td>SNCA: Hs01103383_m1, 18sRNA: Hs03928985_g1, ACTB: Hs01060665_g1, HMBS: Hs00609296, NADH: Hs01072843_m1</td><td>Flurogenic probes</td></tr><tr><td>7500 Fast Real-Time PCR system</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>HEK293T cell line</td><td>ATCC</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>Fetal calf serum</td><td>Biochrom</td><td>S0115</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Biochrom</td><td>A2213</td><td></td></tr><tr><td>Hank&#039;s buffer</td><td></td><td></td><td>5,3 mM KCl, 0.44 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 136.8 mM NaCl pH 6.77, autoclaved.</td></tr><tr><td>DEPC-H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>0.1% diethylpyrocarbonate treated H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>2xBrU-IP Buffer</td><td></td><td></td><td>40 mM Tris-HCl pH 7.5 and 500 mM NaCl in DEPC H2O</td></tr><tr><td>2xBrU-IP+BSA/RNAse inhibitor</td><td></td><td></td><td>2xBrU-IP buffer supplemented with 1 &amp;mu;g/&amp;mu;L BSA and 80 U/mL Ribolock</td></tr><tr><td>BrU-IP+ 1 mg/mL heparin</td><td></td><td></td><td>2xBrU-IP buffer diluted 1:1 DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and supplemented with 1mg/mL heparin</td></tr><tr><td>1xBrU-IP</td><td></td><td></td><td>2xBrU-IP buffer diluted 1:1 in DEPC-H2O and supplemented with 0.5 &amp;mu;g/&amp;mu;L BSA and 20 U/mL Ribolock</td></tr><tr><td>Elution buffer</td><td></td><td></td><td>0.1% SDS in RNAse-free H2O</td></tr></tbody></table>

investigation of rna synthesis using 5-bromouridine labelling and immunoprecipitation

当在两种情况下比较稳态 RNA 水平时, 不可能区分变化是由生产的改变还是 RNA 的退化引起的。该协议描述了一种测量 rna 生产的方法, 使用了 rna 的 5 Bromouridine 标签, 然后是免疫沉淀, 它能够在短时间内对合成的 rna 进行调查 (例如, 1 小时).5-Bromouridine 标签和免疫沉淀在使用有毒转录抑制剂, 如α amanitin 和放线菌 D 的优点是, 在短期使用中, 对细胞的生存能力没有或非常低的影响。然而, 由于 5-Bromouridine-免疫沉淀只捕获在短标签时间内产生的 rna, 缓慢产生, 以及迅速退化的 rna 可能难以测量的方法。由 5-Bromouridine-免疫沉淀捕获的 5-Bromouridine 标记 RNA 可以通过反向转录、定量聚合酶链反应和下一代测序来分析。可以对所有类型的 RNA 进行研究, 该方法不限于本例中所示的测量 mRNA。

我们在 AsPC-1 细胞中进行了老兄标记时间的初步测试。细胞老兄0、1、2、4.5 h, 其次为总 RNA 提取和老兄。GAS5 和 GAPDH 的老兄-IP RNA, 这表明 1 h 标签足以达到约9和44倍的变化相比, GAPDH 和 GAS5 的背景 (0 小时) 分别。
老兄和上述分析被用来研究是否在α synuclein mrna 的稳态水平上发现的类似于马球样激酶 2 (PLK-2) 抑制的变化是由 mRNA 合...

Watch this Scientific Journal Video about Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation at JoVE.com

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

该方法可用于测量 RNA 的合成。5-Bromouridine 被添加到细胞中, 并纳入合成 RNA。rna 的合成是通过标记后立即提取 rna 来测量的, 其次是标记 rna 的 5 Bromouridine 靶向免疫沉淀, 并通过反向转录和定量聚合酶链反应进行分析。

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 ...

investigation-rna-synthesis-using-5-bromouridine-labelling

Universidad San Sebastian

Research

JoVE Journal

Genetics

9.5K 次观看.  Aarhus University. 该方法可用于测量 RNA 的合成。5-Bromouridine 被添加到细胞中, 并纳入合成 RNA。rna 的合成是通过标记后立即提取 rna 来测量的, 其次是标记 rna 的 5 Bromouridine 靶向免疫沉淀, 并通过反向转录和定量聚合酶链反应进行分析。

视频: Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

科研

遗传学

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

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

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

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

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 ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

免疫与感染

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

极快速和特殊的代谢标签的RNA在Vivo与4-Thiouracil （Ers4tU）

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

生物化学

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

生物学

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...