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

Авторы хотели бы поблагодарить д-ра Lan Йи, Института рака в Нью-Джерси, для оказания технической помощи в подготовке ткани корзины. Эта работа финансировалась Национальным институтом здоровья и гранты DK083425 DK070219.

1. Синтез дигоксигенин-11-UTP-Маркированный Riboprobe от ПЦР, созданных с использованием шаблона <ol><li> Для синтеза генов конкретного riboprobe, используйте Entrez Джин (http://www.ncbi.nlm.nih.gov/sites/entrez), чтобы получить ген кДНК ссылка (RefSeq). Используйте Primer3 программы (http://frodo.wi.mit.edu/primer3/) 5 проектировать...

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	<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>

Использование метода, описанного здесь, можно обнаружить мРНК во всех основных типов клеток и тканей отсеков плода мужского и женского мыши ТМП в том числе мезенхимальных колодки, уротелия, гладких мышц, предстательной железы почки, семяизвергательного канала и влагалища. 50 мкм раздел...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th></tr><tr><td> Анти-дигоксигенин антитела, Fab фрагментов </td><td> Roche Applied Science </td><td> 11214667001 </td></tr><tr><td> Блокировка реагента </td><td> Roche Applied Science </td><td> 11096176001 </td></tr><tr><td> Б. М. Фиолетовый AP подложки, осаждая </td><td> Roche Applied Science </td><td> 11442074001 </td></tr><tr><td> Бычьего сывороточного альбумина </td><td> Fisher Scientific </td><td> BP1600-100 </td></tr><tr><td> Клеточные культуры пластины, 24 а </td><td> Гранулирование </td><td> 3524 </td></tr><tr><td> Дигоксигенин 11-UTP </td><td> Roche Applied Science </td><td> 1277073910 </td></tr><tr><td> дНТФ </td><td> Roche Applied Science </td><td> 11969064001 </td></tr><tr><td> Обоюдоострым лезвием бритвы </td><td> Wilkinson Sword </td><td> Классические модели </td></tr><tr><td> Eliminase РНКазы для удаления </td><td> Decon лаборатории </td><td> 1102 </td></tr><tr><td> Формамид </td><td> Сигма </td><td> F5786-1L </td></tr><tr><td> Гель добычи комплект </td><td> Qiagen </td><td> 28704 </td></tr><tr><td> Глутаральдегид, 25% раствор в H 2 O </td><td> Сигма </td><td> G6257-100мл </td></tr><tr><td> Гепарин, натриевая соль </td><td> Сигма </td><td> H3393 </td></tr><tr><td> Перекись водорода, 30% раствор в H 2 O </td><td> Fisher Scientific </td><td> BP2633-500 </td></tr><tr><td> Левамизол </td><td> Сигма </td><td> L9756 </td></tr><tr><td> Loctite 404 Клей быстрого мгновенного набора </td><td> Корпорация Henkel </td><td> 46551 </td></tr><tr><td> Хлорид магния </td><td> Fisher Scientific </td><td> M33-500 </td></tr><tr><td> Малеиновая кислота </td><td> Сигма </td><td> M0375-500G </td></tr><tr><td> Микроцентрифуга труб, 1,5 мл </td><td> Biologix Исследовательская компания </td><td> BP337-100 </td></tr><tr><td> Millicell культуры пластины вставки </td><td> Millipore </td><td> PICM01250 </td></tr><tr><td> Молекулярная шлифовальные смолы </td><td> G-биологических наук </td><td> 786-138PR </td></tr><tr><td> Параформальдегид, 4%-ного раствора в фосфатном буферном растворе </td><td> Affymetrix </td><td> 19943 </td></tr><tr><td> Фосфатным буферным раствором, без Са и Mg </td><td> Депутат Biomedicals </td><td> ICN1760420 </td></tr><tr><td> Полиэфирная сетка, 33 мкм, 12 &quot;х 24&quot; </td><td> Мелкие детали Inc </td><td> CMY-0033-D </td></tr><tr><td> Протеиназы К решению, 20mg/ml </td><td> Amresco </td><td> E195-5мл </td></tr><tr><td> QIAshredder Столбцы </td><td> Qiagen </td><td> 79654 </td></tr><tr><td> Q решение </td><td> Qiagen </td><td> Поставляется с Taq-ДНК-полимеразы </td></tr><tr><td> РНКазы </td><td> Сигма </td><td> R6513 </td></tr><tr><td> РНКазы ингибитор </td><td> Roche Applied Science </td><td> 03335399001 </td></tr><tr><td> RNeasy мини-комплект </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> RNEASY мини-комплект </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> RQ1 РНКазы без ДНКазы </td><td> Promega </td><td> M6101 </td></tr><tr><td> Легкоплавкий легкоплавкой агарозы </td><td> Lonza </td><td> 50101 </td></tr><tr><td> Овцы сыворотке </td><td> Сигма </td><td> S2263-500 мл </td></tr><tr><td> Stericup фильтр, 0.22μm, полиэфирсульфона, 500 мл </td><td> Millipore </td><td> SCGPU05RE </td></tr><tr><td> Азид натрия, гранулированный </td><td> Fisher Scientific </td><td> S227I-100 </td></tr><tr><td> Хлористый натрий </td><td> Fisher Scientific </td><td> BP358-212 </td></tr><tr><td> Додецилсульфата натрия </td><td> Fisher Scientific </td><td> S529-500 </td></tr><tr><td> SSC, 20X решение </td><td> Исследования Products International </td><td> S24022-4000,0 </td></tr><tr><td> Надстрочный III первого прядь синтеза системы </td><td> Invitrogen </td><td> 18080-051 </td></tr><tr><td> Т7 РНК-полимеразы </td><td> Roche Applied Science </td><td> 10881767001 </td></tr><tr><td> Taq-ДНК-полимеразы </td><td> Qiagen </td><td> 201203 </td></tr><tr><td> Трис-HCl </td><td> Fisher Scientific </td><td> BP153-1 </td></tr><tr><td> Твин-20 </td><td> Fisher Scientific </td><td> BP337-100 </td></tr><tr><td> Вибрационный микротома с ванной образца люкс </td><td> Leica Microsystems </td><td> VT1000A </td></tr><tr><td> Дрожжи тРНК </td><td> Roche Applied Science </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

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

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

Здесь мы описываем эффективные высокой пропускной способности<em&gt; На месте</em&gt; Гибридизации (МОГ) метод визуализации моделей экспрессии мРНК в развитии плода разделы предстательной железы мыши ткани. Метод может быть легко адаптирована для визуализации моделей экспрессии мРНК в других тканях, мыши или в тканях, от других видов.

Высокая пропускная способность На месте Гибридизация метод для характеристики Шаблоны экспрессии мРНК в фетальных Мышь Нижняя урогенитального тракта

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

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JoVE Journal

Biology

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

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

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

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

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

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Привет-C: метод изучения трехмерной архитектуры геномов.

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

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

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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. ...

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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 ...

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

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