Name: investigation of rna synthesis using 5-bromouridine labelling and immunoprecipitation
Uploaded: 2018-05-03T13:00:00
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 (D...

<ol>
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Dis. 106, 49-62 (2017).</li><li>Imamachi, N., 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=BRIC-seq:+A+genome-wide+approach+for+determining+RNA+stability+in+mammalian+cells.">BRIC-seq: A genome-wide approach for determining RNA stability in mammalian cells.</a> Methods. 67, 55-63 (2014).</li><li>Jacob, F., Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulatory+mechanisms+in+the+synthesis+of+proteins.">Genetic regulatory mechanisms in the synthesis of proteins.</a> J. Mol. Biol. 3, 318-356 (1961).</li><li>Lee, T. I., Young, R. 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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>&#945;-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 colspan="2">Applied Biosystems</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cell line</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;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&#39;s buffer</td>
			<td></td>
			<td></td>
			<td>5,3 mM KCl, 0.44 mM KH2PO4, 0.2 mM Na2HPO4 and 136.8 mM NaCl pH 6.77, autoclaved.</td>
		</tr>
		<tr>
			<td>DEPC-H2O</td>
			<td></td>
			<td></td>
			<td>0.1% diethylpyrocarbonate treated H2O</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 &#956;g/&#956;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-H2O 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 &#956;g/&#956;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。

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Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

高密度DNA和RNA微阵列.光刻合成、杂交和制备大型核酸库

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...