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>&#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。

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 de Cartagena UDEC

Research

JoVE Journal

Genetics

9.4K 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

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

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 interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

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 ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

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

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

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

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

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

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

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

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

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

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

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

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (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 employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

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

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

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

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable 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 ...