Name: fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo
Uploaded: 2014-10-31T14:00:00
Description: Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the &#8220;priority programmes&#8221; SPP1316 and SPP1617.

1.高产RNA制备<ol><li> RNA分离 注意：高浓度的总RNA，都需要引物延伸反应。离心柱试剂盒通常不产生所需要的RNA的量（〜5 - 16微克在5微升体积）。因此，建议使用酸性硫氰酸胍 - 苯酚 - 氯仿提取法纯化，概述如下。 注：苯酚是致癌物质，有毒和腐蚀性。请阅读材料安全数据表，并在通风橱中使用适当的保护！ <ol><li>生长或治疗细菌细胞（S。在本实施例金黄色葡萄球菌或<em...

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荧光引物延伸是用于确定RNA的5&#39;末端的简单和快速的方法，无论是对TSP-或仲RNA加工鉴定。由于利用荧光引物，所述反应可以建立和无需额外的安全预防措施，运行（不像在壳体的放射性标记的引物）。作为样品，通​​过荧光检测到，它们可以被成像，而在电泳过程中，它允许快速的分析相比，放射性的方法，其中X线片是常用的。 在一般情况下，引物延伸反应的质量在很...

引物延伸1是一个分子的方法来确定特定RNA分子的5&#39;末端到一基座分辨率。其它方法，如5&#39;-RACE（cDNA的末端快速扩增）的优点是快速的周转时间，并很容易地分析的RNA分子的长度不同的混合物的能力。 这种方法的工作原理是使RNA分子利用特异性荧光引物反转录反应，生成一定长度的cDNA片段。这些cDNA分子是在变性聚丙烯酰胺凝胶上运行除了传统Sanger测序反应2，并且可以通过它们的荧光由于使用了荧光标记的引物进行检测。的cDNA片段的长度，然后通过比较来测序梯评估，允许5&#39;核糖核酸端的映射。 传统上，引物延伸反应中结合使用用放射性同位素检测到对X射线胶片的cDNA分子。由于对健康的危害，废物处理问题和易于处理，新的协议利用荧光进行引物延伸用自动测序仪的检测中，尽管其灵敏度稍低。使用荧光标记的引物，由重复的无线标签的过程可以省略，如荧光引物是稳定很长一段时间（超过一年中的手）。 我们在这里描述的方法利用了一种自动凝胶序列，但有轻微的修改，毛细管测序仪还可以用于cDNA的分离和检测3。凝胶分析的并行特性使得它能够检测到少量的RNA切割或加工。另一个优点是该高分辨率这种方法，当终端裂解或者甚至一种碱处理可被检测到。 对于RNA裂解或加工，吨的检测ypically两种不同类型的引物延伸的是有区别的。在一种情况下，酶处理完成后在体外用纯化的RNA和纯化的酶，而在其他情况下，该处理完成的体内 ，将所得的RNA进行纯化。在这两种情况下的RNA，进行引物延伸在体外进行，但视RNA的源上，该方法无论是所谓的体外或体内引物延伸。在我们这里介绍的协议中，我们侧重因为易用性（无纯化的蛋白质需要的话），并有可能在同一时间，以确定转录起始点和处理仅仅对体内引物延伸。然而， 体外引物延伸在原理建立相同的方式，该协议可以作为一个起点。 这里说明的方法可以，只要它们适于高被应用于许多种细菌纯度和高收益的准备核酸。 该研究在我们的实验室着重于毒素抗毒素（TA-）系统4,5，其中，所述引物延伸的方法被广泛使用的字段的调节范围。 TA-系统都存在于原核细胞的基因组，它由一个稳定的和内源性活性的毒性蛋白和大多不稳定的蛋白质或RNA的抗毒素是抵消毒性6,7的小遗传元件。毒素活性有时被抑制复制，细胞壁合成或其他机制通过RNase活性8,9-作用，但是最常见的。典型地，RNA酶的特异性是通过进行不同的测试，其中之一是在引物延伸方法测定的。引物延伸反应是非常适合于该应用程序，如裂解和全长片段的混合物可以同时分析，以确定它们的5&#39;末端。使用体外和体内引物延伸的混合，该具体的毒素RNA酶裂解， 例如，序列特异性，可确定10-13。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> 图的引物延伸步骤（1）概述。细菌培养物，根据实验需要孵育和治疗。总RNA从细胞中，用DNA酶I处理以除去痕量的DNA，并进行使用靶特异性荧光的DNA引物产生的cDNA逆转录反应萃取。基因组DNA或质粒提取并随后用于荧光桑格测序反应中与cDNA片段的大小进行比较。引物延伸产物在变性尿素聚丙烯酰胺凝胶上同时运行桑格测序产物和用自动激光和显微镜分析。测序基础，线与基因带是洛杉矶ST 5&#39;cDNA末端（蓝色箭头）的基地。在菲克特更多信息， 等 。3 <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 整个引物延伸过程的概述可以在图1中找到。简言之，将细菌细胞进行培养，收获，将细胞沉淀溶解和RNA的提取。纯化的RNA，然后用DNA酶I处理以除去的DNA分子，其可作为用于反转录酶的模板的痕迹。特定的荧光引物被加入到RNA的杂交于感兴趣的区域，并随后反向转录，从而产生单链互补DNA（cDNA）。测序梯子是由传统的Sanger测序创建使用荧光引物和分离，旁边的引物延伸cDNA片段的变性聚丙烯酰胺凝胶。由此产生的凝胶是通过比较荧光条带，使所关注的5&#39;端的识别分析。转录的出发点和加工点，然后通过序列比单独评估。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;"><a name="RANGE!A1:D16">Name</a></td>
			<td style="width:196px;">Supplier</td>
			<td style="width:244px;">Catalog Number(s)</td>
			<td style="width:363px;">Comment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">AMV Reverse Transcriptase (20-25 U/&#181;l)</td>
			<td style="width:196px;">NEB / Roche</td>
			<td style="width:244px;">NEB: M0277-T / Roche: 10109118001</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:399px;">DNase I (RNase free)</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2222</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">FastPrep-24 Instrument&#160;</td>
			<td style="width:196px;">MPBio</td>
			<td align="right">116004500</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:399px;">Fluorescently labeled primers</td>
			<td style="width:196px;">Biomers</td>
			<td style="width:244px;">n/a</td>
			<td style="width:363px;">5&#8217; DY-681 modification of &#8220;ordinary&#8221; DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Li-Cor 4200 Sequencer incl. ImagIR Data collection software</td>
			<td style="width:196px;">Li-Cor</td>
			<td style="width:244px;"></td>
			<td style="width:363px;">Product discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">NanoDrop 2000</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Nuclease free water</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM9915G</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Plasmid mini preparation kit</td>
			<td style="width:196px;">QIAGEN</td>
			<td align="right">12125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RapidGel-XL-40% Concentrate</td>
			<td style="width:196px;">USB</td>
			<td style="width:244px;">US75863</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RNA STORAGE BUFFER</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td>AM7000</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Roti-Aqua-P/C/I</td>
			<td style="width:196px;">Carl Roth</td>
			<td style="width:244px;">X985.3</td>
			<td style="width:363px;">Alternative: &#8220;Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)&#8221; from Ambion (AM9720)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">SUPERase&#8226;In RNase Inhibitor</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2696</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP</td>
			<td style="width:196px;">GE Healthcare</td>
			<td style="width:244px;">RPN2538</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">TRIzol reagent</td>
			<td style="width:196px;">life technologies</td>
			<td style="width:244px;">15596-026</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Zirconia/Silica Beads 0.1 mm</td>
			<td style="width:196px;">BioSpec</td>
			<td style="width:244px;">11079101z</td>
			<td style="width:363px;"></td>
		</tr>
	</tbody>
</table>

fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo

基于荧光的引物延伸（FPE）是一个分子的方法来确定转录起始点或RNA分子的加工位点。这是通过使用特定的荧光标记的引物，将所得cDNA片段通过变性聚丙烯酰胺凝胶电泳随后分析感兴趣的RNA的反转录来实现的。同时，传统的Sanger测序反应是在凝胶上，以该cDNA片段的末端映射到它们的确切相应的碱。而相比之下，5&#39;-RACE（cDNA末端的快速扩增），其中，所述产品必须被克隆和测序的多个候选，堆积由引物延伸所产生的cDNA片段可以同时在一个凝胶运行检测。此外，整个过程（从反转录到的结果的最终分析）可以在一个工作日内完成。通过使用荧光标记的引物，使用危险的放射性同位素标记的试剂可避免与处理时间缩短为可以在电泳过程中，被检测的产品。 在下面的协议中，我们描述了一种在体内的荧光引物延伸的方法来可靠地和快速地检测RNA的5&#39;末端推断的转录起始点和RNA加工位点（ 例如，通过毒素-抗毒素系统组件）的S.金黄色葡萄球菌，大肠杆菌大肠杆菌和其它细菌。

如示于图6中 ，引物延伸反应可以用来确定感兴趣转录物的转录起点，并可以帮助推断启动子区域（典型地由在-10和-35元件识别）。最上面的（最长）的cDNA片段代表了mRNA的5&#39;端，因此可相比的测序梯容易地进行映射。 <img alt="图6" src="/files/ftp_upload/52134/52134fig6highres.jpg" /> 引物延伸反应的图6。代表性结果 。左边一个完整的活体引物延伸，...

Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

基于荧光引物延伸法确定转录起点和核糖核酸酶的切割位点<em&gt;在体内</em

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Research

JoVE Journal

Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Human ES cells: Starting Culture from Frozen Cells

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

科研

生物学

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis Extension Response PER Assay in Drosophila

长鼻扩展响应（PER）的含量在果蝇

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

引物延伸捕获：从严重退化的DNA来源有针对性的顺序检索

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...