这项工作得到了美国国立医学研究所 (NIH (P20GM103499).) 的 INBRE 赠款的支持, 并资助 CA555536 & CA154664 从国家癌症研究所到 P.H.H。这一方案得到部分支持, 从霍华德休斯医学院通过大学预科 & 本科生科学教育项目, 并由国家研究资源中心 (5 P20 RR016461) 赠款。和国立卫生研究院 (8 P20 GM103499)。

1. 培养和代谢标记
<ol><li>一次戳一个无菌1.5 毫升微离心管的中心, 用18口径的针头, 允许适当的曝气。接种500µL 的补充无菌7H9 汤与5µL 的m 耻从一夜成名的文化增长12。 注: 一公升7H9 汤的配方为: 4.7 克商业7H9 粉, 2 毫升甘油, 1 毫升吐温 80, 0.85 克氯化钠, 5 克白蛋白, 2 g 葡萄糖, H2O (使总体积到 1 L)。<ol>
<li>
(警告)按照标准的防护做法, 使用20µCi/mL 的 [32P] Na2HPO4来对区域性媒体进行尖峰。生长在37° c 和 1200 rpm 搅拌的细菌, 在一个9毫米厚的亚克力盾牌后面的振动筛孵化器。</li>
	</ol>
</li>
	<li>在 midlog 阶段 (外径 600 nm ~ 0.5), 将整个培养物转化为2毫升的螺旋帽管, 室温下的颗粒状放射性细菌离心为2分钟, 在 1万 x g。适当的废物容器。</li>
</ol>2. 总 rna 的制备
注: 根据制造商的协议, 使用商业 rna 提取试剂 (含硫氰酸胍、苯酚和氯仿) 从细菌小球中提取总 rna。
<ol><li>添加1毫升的商业 RNA 提取试剂和大约200µL 玻璃珠上的颗粒。在最大搅拌的情况下, 将管帽安全地盖在一个均质器内, 并将细菌扰乱2分钟 (在选定的仪器上设置编号五 (请参见材料表))。</li>
	<li>离心机样品在 1.2万 x g 为10分钟在4° c。将上清液转移到新的2毫升管, 并适当地丢弃珠子。十五年代用手用力加0.2 毫升氯仿和摇管. 离心机在 1.2万 x g 为15分钟在4° c。</li>
	<li>通过在45°上钓鱼, 将样品的水相收集到一个新的管子中。避免绘制任何界面或有机层。添加2µL 色沉淀和0.5 毫升100% 异丙醇。用手用力摇动管子5° c 1.2万 x g 的离心机10分钟。</li>
	<li>从管中取出上清液, 并适当地丢弃, 因为液体馏分可能含有放射性的痕迹。空气干燥颗粒为5分钟, 然后重在200µL 2X 生理盐水-柠檬酸钠 (SSC), 0.1% (瓦特/五) 十二烷基硫酸钠 (SDS) 决赛。 注:20 X SSC 的成份为3.0 米氯化钠, 0.3 米柠檬酸钠, pH 值7.0。</li>
</ol>3. 96 井印版的研制
注: tRNA 微阵列是手工打印的四十二70的 DNA 寡核苷酸互补的 3 ' 结束的每个m. 耻tRNA (终端共同国家评估排除) 使用八针机器人。DNA 寡核苷酸探针的序列和探针布局在 well-plate 和阵列被提供 (补充文件 1-板料1到3分别)。
<ol><li>设计 DNA 探针首先检索 tRNA 序列或 tRNA 编码基因从 tRNA 数据库, 如基因组 tRNA 数据库13。修剪保守3的最终评估, 如果编码。反向补充结果序列。基于前70核苷酸的顺序探针。</li>
	<li>重干 DNA 探针在水中。将探头浓度调整到100µM。在 96 well-plate, 准备120µL 50 µM 寡在 3X ssc, 0.01% (瓦特/v) sds 决赛 (稀释60µL 的股票探头在个人井与60µL 6X ssc, 0.01% (瓦特/v) sds。</li>
	<li>盖板与锡纸, 以防止蒸发或污染, 立即冻结, 并保持在-20 ° c, 直到需要。</li>
</ol>4. 阵列打印
<ol><li>在室温下解冻96井板 (大约需要20分钟)。</li>
	<li>用钻石笔将胺涂层玻璃贴上标签。将幻灯片放入索引单元。跟随网格安置并且做如下。<ol>
<li>小心地将复制器针脚浸入井中。</li>
		<li>轻轻地在玻璃上打印阵列, 使用最小的压力 (你会感觉到小的阻力)。继续打印, 不清洗机器人, 直到完成与块 A。</li>
	</ol>
</li>
	<li>当准备移动到下一个街区时, 按照下面描述的洗涤程序。<ol>
<li>在5% 漂白和轻轻摇动的倾角复制器。</li>
		<li>按复制器进入吸水纸。</li>
		<li>将复制体浸在蒸馏水中, 轻轻摇动, 然后按入纸。重复此步骤一次。</li>
		<li>在异丙醇上蘸复制器, 轻轻摇动, 然后按进纸张。</li>
		<li>将复制器在风扇上晾干大约二十年代, 然后再移动到96井板的下一个区块。继续打印所需的数量。</li>
	</ol>
</li>
	<li>允许幻灯片晾干, 然后用 254 nm 的 UV 交将寡与幻灯片交联。<ol>
<li>将能量级别设置为999990µJ/cm2。</li>
		<li>将幻灯片正面放在交的干净表面上。按 "开始"。</li>
	</ol>
</li>
	<li>在磁搅拌器上, 在室温和慢搅拌下, 用450毫升的堵塞溶液在一夜之间阻挡阵列。收集和重用阻塞解决方案最多四次。 注: 堵塞解决方案的配方是: 125 mm 磷酸盐缓冲液 (NaH2PO4/Na2HPO4) pH 7.2, 2.25% SDS (含/五), 0.5 毫米 EDTA, 0.5X SSC, 1% BSA (w/v)。</li>
	<li>在500毫升蒸馏水中, 在室温下5分钟洗涤2次幻灯片。用微阵列离心机将十年代的离心在室温下干燥。存储阵列在干燥和黑暗的地方长达6月。</li>
</ol>5. 阵列杂交
<ol><li>在杂交前, 用沸水冲洗滑块, 用离心法干燥 (如 4.6)。</li>
	<li>放置阵列内的杂交盒和负载标记 RNA 样本。</li>
	<li>运行自动杂交洗站的最大再现性。<ol>
<li>程序步骤如下。条件垫片为2分钟, 在75° c。在60° c 时引入探头。 变性探针和样品5分钟在90° c。杂交样品为3小时在60° c。<ol>
<li>在50° c 下两次冲洗幻灯片, 2X ssc, 0.1% (带 v) SDS, 十年代和二十年代举行. 在42° c 两次洗涤幻灯片与 0.1X ssc, 0.1% SDS, 流动十年代和持有二十年代. 在42° c 两次清洗幻灯片 0.1 X ssc, 流动十年代并且举行二十年代。</li>
		</ol>
</li>
	</ol>
</li>
	<li>离心干燥的幻灯片 (如 4.6)。</li>
</ol>6. 阵列量化
<ol><li>用薄薄的塑料薄膜包装幻灯片。在最敏感的设置上使用盖革计数器检查放射性信号的幻灯片。根据信号强度, 将幻灯片暴露在室温为10至90小时的曝光盒中的存储荧光屏上。 注意: 由于盖革计数器的灵敏度因仪器而异, 因此必须根据经验对曝光时间进行优化。</li>
	<li>使用 phosphorimager 扫描50µm 分辨率的幻灯片。使用微阵列探查器升级的免费图像 J 软件, 在每个探针点量化和背景减去放射性强度。</li>
	<li>使用电子表格组织和处理数据。使用条件格式工具生成热映射。例如代表每个探针信号作为分数 (用‰表示) 总探针信号。</li>
</ol>

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没有利益冲突宣布。

β粒子的排放是已知的抑菌和杀菌剂14, 但是在测试范围内的辐射对细胞的适应性没有显著影响。闪烁计数表明, 放射性被纳入25% 的总细胞提取物, 随后, 1% 的纯化总 rna。
探测器的大小相同, 熔点和 GC 含量相同, 同时 macroarray 斑点、杂交和定量的统一协议允许直接从 spot 信号中测量 tRNA 水平。
现货是可重现和具体的。它的大动态范围和易于调整的门槛允许分析低丰富的物种, 如转运与聚, 只需延长阵列曝光时间9。在对所需设备进行初步投资后, 每个样品的运行费用, 包括消耗品, 平均每样15美元。
斑点适用于任何生物体, 其基因组可用于探针设计。在体外生长的模型生物体是新陈代谢标记的理想候选者。由于哺乳动物的基因组经常编码许多 isodecoders (转运, 共享相同的 anticodons, 但显示在他们的身体序列的细微差异) 探针需要部分退化, 以保存接近 tRNA 物种的同源杂交。最后, 与悬浮物中生长的生物体相比, 附着在哺乳动物细胞中的总 RNA 的数量要低得多, 例如m 耻, 所以文化需要大幅度地扩大。

细胞 tRNA 水平的波动根据条件, 如细胞类型, 环境, 和压力1,2,3。现场, 简化的观察 tRNA 的平台, 是一个原始的, 可靠的, 直接的技术, 允许快速和精确的量化的转移 RNA 水平的实验室培养的有机体。
转运具有显著稳定的二级和三级结构4。它们还显示许多后修改5。这些特征代表重要的结构和序列路障偏置, 或有时阻止使用标准的基准 RNA 量化技术6进行直接和可重现的 tRNA 分析。世界各地的研究小组被迫发展创造性的, 但往往是复杂的技术来评估细胞 tRNA 水平。其中的一些方法包括: (1) two-dimensional 电泳分离新陈代谢标记转运结合有条理的斑点分配的北印迹1;(2) 使用经过仔细标准化的放射性 DNA 探针的北印迹的个体量化7;(3) 蒜标记与菁荧光其次是微阵列分析3, 和 (4)体外剥离 tRNA 修饰使用重组 demodification 酶结合反转转录和随后高通量排序8。
斑点是两个标准和直接的方法的简单和原始的组合: (1) RNA 身体标记, 包括在生长的有机体, 放射性总 rna 和 (2) tRNA macroarrays, 系统和微型基因组工具优化 tRNA 隔离。
样品从活有机体被简化到定量平台。他们是直接分析后提取, 而不是进一步处理, 大大限制了偏见相比, 需要蒜放大或酶治疗技术。斑点是多才多艺的, 可以很容易地结合到 polysome 分馏, 以确定和量化 tRNA 亚群, 与翻译核糖体的物理联系。
此处介绍的协议针对分枝杆菌耻进行了优化, 并且还成功地用于在大肠杆菌9、酿酒酵母10、鼠标和人类细胞培养中对转运进行剖面分析11.

<table><tbody><tr><td>Glass slide indexing system</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 470</td><td></td></tr><tr><td>Glass slide arrayer replicator</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 478</td><td></td></tr><tr><td>96 well microplate indexing system</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 472</td><td></td></tr><tr><td>Wash and blot station</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 475</td><td></td></tr><tr><td>Replicator pin dryer</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 474</td><td></td></tr><tr><td>Amine coated slides</td><td>Molecular Devices</td><td>K2615</td><td></td></tr><tr><td>Hybridizer / Automated hybridization and washing station</td><td>Digilab</td><td>Hyb10022</td><td></td></tr><tr><td>Shaker incubator</td><td>Eppendorf Themomixer</td><td>05-400-200</td><td></td></tr><tr><td>Screw cap 2 ml tubes</td><td>Thermo Scientific</td><td>21-403-201</td><td></td></tr><tr><td>[32P] Na2HPO4</td><td>Perkin Elmer</td><td>NEX011001MC</td><td></td></tr><tr><td>Guanidinium thiocyanate-phenol-chloroform (Trizol)</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>0.5 mm glass beads</td><td>Research Products International Corp.</td><td>9831</td><td></td></tr><tr><td>Mini bead mill</td><td>VWR</td><td>25-020</td><td></td></tr><tr><td>Storage phosphore screen</td><td>Fujifilm</td><td>BAS-IP SR 0813</td><td></td></tr><tr><td>Phosphorimager</td><td>GE Healthcare</td><td>Typhoon FLA7000</td><td></td></tr><tr><td>DNA oligonucleotides</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>UV crosslinker</td><td>Spectroline</td><td>Select series 254 nm</td><td></td></tr><tr><td>Microarray centrifuge</td><td>Arrayit Corporation</td><td>MHC110V</td><td></td></tr><tr><td>&lt;em&gt;Mycobacterium smegmatis&lt;/em&gt;</td><td>ATCC</td><td>700084</td><td></td></tr><tr><td>Single-use needles</td><td>BD (Becton Dickinson)</td><td>305185</td><td></td></tr><tr><td>2 ml centrifuge tube</td><td>Fisherbrand</td><td>14-666-317</td><td></td></tr><tr><td>Precipitant (GlucoBlue)</td><td>Invitrogen</td><td>AM9516</td><td></td></tr><tr><td>7H9 broth</td><td>Difco</td><td>271310</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Chemicals</td><td>C298-500</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemicals</td><td>A451-4</td><td></td></tr><tr><td>20 X SSC</td><td>Lonza</td><td>51205</td><td></td></tr><tr><td>SDS</td><td>Fisher Bioreagents</td><td>BP166-100</td><td></td></tr><tr><td>hybridisation cassette</td><td>GE Healthcare</td><td>63-0035-44</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Tween 80</td><td>Amresco</td><td>M126-100ML</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Albumin</td><td>Spectrum Chemicals</td><td>A3611</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Chemicals</td><td>D16-1</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic Acid)</td><td>Fisher Bioreagents</td><td>BP2482-500</td><td></td></tr><tr><td>0.5 M Phosphate buffer pH 7.2</td><td>Alfa Aesar</td><td>AAJ63974AP</td><td></td></tr></tbody></table>

metabolic labeling and profiling of transfer rnas using macroarrays

转移 rna (tRNA) 是丰富的短编码 RNA 种类, 通常是76到90核苷酸的长度。转运直接负责蛋白质的合成, 将密码的 mRNA 转化为氨基酸序列。长久以来, 转运被认为是缺乏监管功能的内部分子。然而, 越来越多的证据表明, 细胞 tRNA 水平在对应的变化条件, 如细胞类型, 环境, 和压力波动。tRNA 表达的波动直接影响基因的翻译, 有利于或抑制特定蛋白质的表达。最终理解蛋白质合成的动态需要开发能够提供高质量的 tRNA 剖面的方法。我们这里提出的方法被命名为 SPOt, 它代表了用于观察 tRNA 的流线型平台。斑点由三步开始以细胞培养的新陈代谢的标记与放射性磷酸盐, 其次胍硫氰酸盐-苯酚-氯仿提取放射性总 rna 和最后杂交在 in-house 打印macroarraystRNA 水平的估计, 量化的放射性强度在每个探针点。在这里提出的协议中, 我们在分枝杆菌耻mc2155 转运, 一个致病细菌经常被用作模型生物体来研究结核病。

三独立的生物复制表明, 在测试的生长条件下, 所有的m 耻转运都表示为高于背景级别 (图 1)。在这个特定的实验中, 观察到的每个探针的标准偏差包括 2% (Arg) 和 22% (胱氨酸 (配合力)), 中间值为 5% (图 1)。复制之间的假变化很大程度上受到阵列准备和杂交等因素的影响。高再现性是通过仔细和一致的阵列打印以及自动样品杂交实现的。tRNA 与 tRNA (TAC) 之间有5倍的差异, 分别是最高和最低的丰种。斑点显示 tRNA isoacceptors 的表达不均匀。Isoacceptors 是种, 持有相同的氨基酸, 但显示不同的子序列, 因此解码不同的密码在基因。例如, 所有丙氨酸 isoacceptors 在相似的水平被表达, 而最高和最低的精氨酸接受转运不同由3倍。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56898/56898fig1.jpg"> 图 1: 代表 tRNA macroarray 结果.(A)扫描细菌、鼠标和人类 macroarrays。m 耻数组只使用43探针, 而不是48只用于鼠标和人。因此, 细菌阵列显示40个空点 (每个阵列上的探针被复制八次), 与背景相混合。他们中的大多数都聚集在左上角。(B)在最佳增长条件下的m 耻中的 tRNA 配置文件的直方图和图表示形式。探针信号是平均的三独立实验 (包括细菌生长, 新陈代谢的标记, 提取和阵列杂交) 和被表达作为每一千价值总转运。三复制的标准偏差分别表示为误差线和橙色在直方图和图上的深浅。<a href="http://cloudflare.jove.com/files/ftp_upload/56898/56898fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays at JoVE.com

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

我们描述了一个原始的传输 RNA 分析平台名为 SPOt (简化的观察 tRNA 平台)。斑点同时测量所有转运在生物样品中的细胞水平, 仅三步, 并在不到24小时。

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

metabolic-labeling-and-profiling-of-transfer-rnas-using-macroarrays

Research

JoVE Journal

Genetics

5.8K 次观看.  College of Charleston. 我们描述了一个原始的传输 RNA 分析平台名为 SPOt (简化的观察 tRNA 平台)。斑点同时测量所有转运在生物样品中的细胞水平, 仅三步, 并在不到24小时。

视频: Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

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 ...

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 ...

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

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

医学

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

生物学

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

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 ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
</div>
<div class="chapter_text" id="script_2">
The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
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In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
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Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
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You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

代谢标记

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

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

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

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

在乳腺癌细胞雌激素调节小分子RNA的剖析

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

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

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

代谢标记

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

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

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

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

在乳腺癌细胞雌激素调节小分子RNA的剖析

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

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

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

代谢标记

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心