Name: a high throughput in situ hybridization method to characterize mrna expression patterns in the fetal mouse lower urogenital tract
Uploaded: 2011-08-19T13:00:00
Description: Watch this Scientific Journal Video about A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract at JoVE.com

作者想感谢的技术援助，准备组织筐兰一，新泽西癌症研究所，博士。这项工作是由美国国立卫生赠款DK083425和DK070219研究院。

1。地高辛- 11 - UTP标记Riboprobe从一个PCR生成的模板合成<ol><li>为了合成一个特定基因riboprobe，使用Entrez基因（http://www.ncbi.nlm.nih.gov/sites/entrez）获得的基因的cDNA参考序列（的RefSeq） 。使用Primer3程序（http://frodo.wi.mit.edu/primer3/）5，设计基因特异性PCR引物对cDNA序列的3&#39; -地区。 PCR引物的选择推荐的参数描述（http://www.gudmap.org/Research/Protocols/Vezina/Riboprobe_Syn.html）。 </li><li>使用MegaBLAST?...

<ol>
	<li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+possible+influence+of+temporal+factors+in+androgenic+responsiveness+of+urogenital+tissue+recombinants+from+wild-type+and+androgen-insensitive+(Tfm)+mice.">The possible influence of temporal factors in androgenic responsiveness of urogenital tissue recombinants from wild-type and androgen-insensitive (Tfm) mice.</a> Journal of Experimental Zoology. 205, 181-181 (1978).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal,+cellular,+and+molecular+regulation+of+normal+and+neoplastic+prostatic+development.">Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.</a> Journal of Steroid Biochemistry and Molecular Biology. 92, 221-221 (2004).</li><li>Price, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+development+and+structure+in+the+prostate.">Comparative aspects of development and structure in the prostate.</a> National Cancer Institute Monographs. 12, 1-1 (1963).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+interactions--I.+Induction+of+prostatic+phenotype+in+urothelium+of+testicular+feminized+(Tfm/y)+mice.">Stromal-epithelial interactions--I. Induction of prostatic phenotype in urothelium of testicular feminized (Tfm/y) mice.</a> Journal of Steroid Biochemistry. 14, 1317-1317 (1981).</li><li>Rozen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3+on+the+WWW+for+general+users+and+for+biologist+programmers.">Primer3 on the WWW for general users and for biologist programmers.</a> Methods in Molecular Biology. 132, 365-365 (2000).</li><li>Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+greedy+algorithm+for+aligning+DNA+sequences.">A greedy algorithm for aligning DNA sequences.</a> Journal of Computational Biology. 7, 203-203 (2000).</li><li>Vezina, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dioxin+causes+ventral+prostate+agenesis+by+disrupting+dorsoventral+patterning+in+developing+mouse+prostate.">Dioxin causes ventral prostate agenesis by disrupting dorsoventral patterning in developing mouse prostate.</a> Toxicological Sciences. 106, 488-488 (2008).</li><li>Wilkinson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+messenger+RNA+by+in+situ+hybridization+to+tissue+sections+and+whole+mounts.">Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.</a> Methods in Enzymology. 225, 361-361 (1993).</li></ol>

使用这里介绍的方法，它可以检测到在所有主要类型的细胞和胎儿的雄性和雌性小鼠的LUT，包括间质垫，尿路上皮，平滑肌，前列腺芽，射精管，及阴道组织的车厢的mRNA。在本议定书中所使用的50μm的部分有足够厚，以解决组织架构（如血管）的优势，但足够薄，以避免探头诱捕，这是一个方法论的问题，在整个安装ISH普遍遭遇。每一个新的riboprobe评估阳性对照组织，染色已在先前发表的研究报?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>抗地高辛抗体Fab片段</td><td>罗氏应用科学部</td><td> 11214667001 </td></tr><tr><td>阻断试剂</td><td>罗氏应用科学部</td><td> 11096176001 </td></tr><tr><td> BM紫美联社衬底，沉淀</td><td>罗氏应用科学部</td><td> 11442074001 </td></tr><tr><td>牛血清白蛋白</td><td> Fisher Scientific则</td><td> BP1600 - 100 </td></tr><tr><td>细胞培养板，以及24 </td><td>康宁</td><td> 3524 </td></tr><tr><td>地高辛11 -双绞线</td><td>罗氏应用科学部</td><td> 1277073910 </td></tr><tr><td> dNTPs </td><td>罗氏应用科学部</td><td> 11969064001 </td></tr><tr><td>双刃刀片</td><td>威尔金森剑</td><td>经典模型</td></tr><tr><td> Eliminase核糖核酸卸妆</td><td> DECON实验室</td><td> 1102 </td></tr><tr><td>甲酰胺</td><td>西格玛</td><td> F5786 - 1L </td></tr><tr><td>凝胶提取试剂盒</td><td> Qiagen公司</td><td> 28704 </td></tr><tr><td>戊二醛，25％的H 2 O的解决方案</td><td>西格玛</td><td> G6257 - 100ML </td></tr><tr><td>肝素，钠盐</td><td>西格玛</td><td> H3393 </td></tr><tr><td>过氧化氢 ​​，30％的溶液中H 2 O </td><td> Fisher Scientific则</td><td> BP2633 - 500 </td></tr><tr><td>左旋咪唑</td><td>西格玛</td><td> L9756 </td></tr><tr><td>乐泰404快速设置瞬间粘合剂</td><td>汉高公司</td><td> 46551 </td></tr><tr><td>氯化镁</td><td> Fisher Scientific则</td><td> M33 - 500 </td></tr><tr><td>马来酸</td><td>西格玛</td><td> M0375 - 500G </td></tr><tr><td>离心管，1.5ml的</td><td> Biologix研究公司</td><td> BP337 - 100 </td></tr><tr><td> Millicell小文化板插入</td><td> Millipore公司</td><td> PICM01250 </td></tr><tr><td>分子研磨树脂</td><td> G - Biosciences公司</td><td> 786 - 138PR </td></tr><tr><td>多聚甲醛，4％的磷酸盐溶液缓冲液</td><td> Affymetrix公司</td><td> 19943 </td></tr><tr><td>磷酸盐缓冲液，无钙和镁</td><td> MP生物医学</td><td> ICN1760420 </td></tr><tr><td>聚酯网，33微米，12“× 24” </td><td>小零件公司</td><td> CMY - 0033 - D </td></tr><tr><td> 20mg/ml蛋白酶K溶液， </td><td> AMRESCO </td><td> E195 - 5ML </td></tr><tr><td> QIAshredder列</td><td> Qiagen公司</td><td> 79654 </td></tr><tr><td> Q解决方案</td><td> Qiagen公司</td><td>提供Taq DNA聚合酶</td></tr><tr><td>核糖核酸酶</td><td>西格玛</td><td> R6513 </td></tr><tr><td> RNase抑制剂</td><td>罗氏应用科学部</td><td> 03335399001 </td></tr><tr><td>的RNeasy微型试剂盒</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td>的RNeasy微型试剂盒</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td> RQ1 RNase的免费DNA酶</td><td> Promega公司</td><td> M6101 </td></tr><tr><td> SeaPlaque低熔化琼脂糖</td><td>龙沙</td><td> 50101 </td></tr><tr><td>羊血清</td><td>西格玛</td><td> S2263 - 500ML </td></tr><tr><td> Stericup过滤装置，0.22μm的，聚醚砜，500ML </td><td> Millipore公司</td><td> SCGPU05RE </td></tr><tr><td>叠氮化钠，呈颗粒状</td><td> Fisher Scientific则</td><td> S227I - 100 </td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> BP358 - 212 </td></tr><tr><td>十二烷基硫酸钠</td><td> Fisher Scientific则</td><td> S529 - 500 </td></tr><tr><td>南南合作，20X解决方案</td><td>研究产品国际</td><td> S24022 - 4000.0 </td></tr><tr><td>标III第一链合成系统</td><td> Invitrogen公司</td><td> 18080-051 </td></tr><tr><td> T7 RNA聚合酶</td><td>罗氏应用科学部</td><td> 10881767001 </td></tr><tr><td> Taq DNA聚合酶</td><td> Qiagen公司</td><td> 201203 </td></tr><tr><td>的Tris - HCl </td><td> Fisher Scientific则</td><td> BP153 - 1 </td></tr><tr><td>吐温20 </td><td> Fisher Scientific则</td><td> BP337 - 100 </td></tr><tr><td>与豪华标本浴振动切片机</td><td>徕卡显微系统</td><td> VT1000A </td></tr><tr><td>酵母tRNA </td><td>罗氏应用科学部</td><td> 109495 </td></tr></tbody></table>

a high throughput in situ hybridization method to characterize mrna expression patterns in the fetal mouse lower urogenital tract

较低的泌尿生殖道（LUT）的发展是一个复杂的的过程。这种复杂性体现在从依赖雄激素信号和上皮-间质相互作用1,2胎儿男性尿道，前列腺的形成。了解前列腺发育的分子机制，可能揭示的增长是不恰当地从沉睡中苏醒过来，在以后的生活会引起良性前列腺增生和前列腺癌等前列腺疾病的机制。 发展LUT的解剖结构复杂。 16.5天的概念（DPC）的时间前列腺萌芽开始，许多类型的细胞存在。血管，神经和平滑肌驻留在间质的基质 3 。这间质围绕着一个多层的上皮细胞和引起胎儿通过雄激素受体 ​​依赖旁分泌信号 4前列腺。身份的基质雄激素受体反应的前列腺癌的发展，针对这些基因的前列腺导管上皮形式尚不完全清楚的机制所需基因。能够准确地识别细胞类型和本地化内表达的具体因素是要进一步了解前列腺癌的发展。原位杂交（ISH）允许一个组织内mRNA的本地化。因此，这种方法可用于识别信号分子及其受体的表达模式和时间，从而阐明潜在的前列腺发育调节。 在这里，我们描述了一种高通量的原位杂交技术来识别在胎鼠LUT的使用振动切片机切段mRNA的表达模式。这种方法提供了比其他ISH的协议几个优点。执行上坚持一个幻灯片的超薄切片的原位杂交是技术上的困难;冰冻切片经常有结构质量差而冰冻切片和石蜡切片，往往会造成弱信号的分辨率。执行整装组织的ISH，可能会导致探头捕获。相比之下，我们的高通量技术采用厚切的部分，显示详细的组织架构。修改离心管允许在原位杂交的过程中容易处理的部分。 4 mRNA转录的一个最多可从一个单一的LUT高达24 mRNA的单次运行中检测到的成绩单17.5dpc筛选，从而降低成本和最大限度地提高效率。这种方法允许多个治疗组相同，并作为一个单元处理，从而消除任何解释数据的偏差。大多数有针对性地对前列腺的研究人员，这种方法提供了一个在胎鼠尿道前列腺导管网络，使人们产生低和高丰度的mRNA转录的空间和时间的位置。

Watch this Scientific Journal Video about A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract at JoVE.com

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

在这里，我们描述了一个高效的高吞吐量<em&gt;原位</em&gt;杂交（ISH）可视化mRNA的表达模式，在胎鼠的前列腺组织切片的方法。该方法可以很容易地适应可视化，在其他的鼠标组织或来自其他物种的组织的mRNA的表达模式。

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Research

JoVE Journal

Biology

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

A high-throughput method to globally study the organelle morphology in S. cerevisiae

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...