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

लेखकों के लिए तकनीकी सहायता के लिए ऊतक टोकरी तैयारी में डा. Lan यी, न्यू जर्सी के कैंसर संस्थान, शुक्रिया अदा करना चाहूँगा. यह काम स्वास्थ्य DK083425 और DK070219 अनुदान के राष्ट्रीय संस्थान द्वारा वित्त पोषित किया गया था.

1. पीसीआर उत्पन्न टेम्पलेट से Digoxigenin-11-UTP लेबल Riboprobe के संश्लेषण <ol><li> एक जीन विशिष्ट riboprobe synthesize करने के लिए, Entrez जीन (http://www.ncbi.nlm.nih.gov/sites/entrez) का उपयोग करने के लिए जीन सीडीएनए संदर्भ अनुक्रम (RefSeq) प्राप्त. सीडीएनए अनुक्रम ?...

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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>

पद्धति का उपयोग करके यहाँ वर्णित है, यह संभव है के सभी प्रमुख प्रकार की कोशिकाओं और भ्रूण पुरुष और महिला mesenchymal पैड, urothelium, चिकनी पेशी, prostatic कलियों, उद्गार वाहिनी, और योनि सहित माउस LUTs के ऊतक डिब्बों में mRNAs का पत?...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th></tr><tr><td> विरोधी Digoxigenin एंटीबॉडी, फैब टुकड़े </td><td> Roche एप्लाइड साइंस </td><td> 11214667001 </td></tr><tr><td> अभिकर्मक अवरुद्ध </td><td> Roche एप्लाइड साइंस </td><td> 11096176001 </td></tr><tr><td> बी.एम. बैंगनी एपी सब्सट्रेट precipitating, </td><td> Roche एप्लाइड साइंस </td><td> 11442074001 </td></tr><tr><td> गोजातीय सीरम albumin </td><td> फिशर साइंटिफिक </td><td> BP1600-100 </td></tr><tr><td> सेल संस्कृति प्लेट, अच्छी तरह से 24 </td><td> Corning </td><td> 3524 </td></tr><tr><td> 11-UTP Digoxigenin </td><td> Roche एप्लाइड साइंस </td><td> 1277073910 </td></tr><tr><td> dNTPs </td><td> Roche एप्लाइड साइंस </td><td> 11969064001 </td></tr><tr><td> डबल धार धार </td><td> Wilkinson तलवार </td><td> क्लासिक मॉडल </td></tr><tr><td> Eliminase RNase हटानेवाला </td><td> Decon प्रयोगशालाओं </td><td> 1102 </td></tr><tr><td> Formamide </td><td> सिग्मा </td><td> F5786-1L </td></tr><tr><td> जेल निकासी किट </td><td> Qiagen </td><td> 28704 </td></tr><tr><td> Glutaraldehyde, एच 2 हे में 25% समाधान </td><td> सिग्मा </td><td> G6257-100ml </td></tr><tr><td> हेपरिन, सोडियम नमक </td><td> सिग्मा </td><td> H3393 </td></tr><tr><td> हाइड्रोजन पेरोक्साइड, एच 2 हे में 30% का समाधान </td><td> फिशर साइंटिफिक </td><td> BP2633-500 </td></tr><tr><td> Levamisole </td><td> सिग्मा </td><td> L9756 </td></tr><tr><td> लॉकटाइट 404 जल्दी सेट तुरंत चिपकने वाला </td><td> हेंकेल कॉर्प </td><td> 46551 </td></tr><tr><td> मैग्नीशियम क्लोराइड </td><td> फिशर साइंटिफिक </td><td> M33-500 </td></tr><tr><td> Maleic एसिड </td><td> सिग्मा </td><td> M0375-500G </td></tr><tr><td> Microcentrifuge ट्यूबों, 1.5mL </td><td> Biologix रिसर्च कंपनी </td><td> BP337 - 100 </td></tr><tr><td> Millicell संस्कृति थाली डालने </td><td> Millipore </td><td> PICM01250 </td></tr><tr><td> आण्विक पीस राल </td><td> G-बायोसाइंसेज </td><td> 786-138PR </td></tr><tr><td> Paraformaldehyde, फॉस्फेट में 4% समाधान खारा buffered </td><td> Affymetrix </td><td> 19943 </td></tr><tr><td> फास्फेट खारा Ca और मिलीग्राम के बिना, buffered </td><td> सांसद Biomedicals </td><td> ICN1760420 </td></tr><tr><td> पॉलिएस्टर जाल, 33 माइक्रोन, &quot;x 24&quot; 12 </td><td> लघु पार्ट्स इंक </td><td> CMY-0033-D </td></tr><tr><td> Proteinase कश्मीर समाधान, 20mg/ml </td><td> Amresco </td><td> E195 - 5ml </td></tr><tr><td> QIAshredder स्तंभ </td><td> Qiagen </td><td> 79654 </td></tr><tr><td> क्यू समाधान </td><td> Qiagen </td><td> Taq डीएनए पोलीमरेज़ साथ परंतु </td></tr><tr><td> RNase </td><td> सिग्मा </td><td> R6513 </td></tr><tr><td> RNase अवरोध करनेवाला </td><td> Roche एप्लाइड साइंस </td><td> 03335399001 </td></tr><tr><td> RNeasy मिनी किट </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> RNEASY मिनी किट </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> RQ1 RNase मुक्त DNase </td><td> Promega </td><td> M6101 </td></tr><tr><td> SeaPlaque कम पिघल agarose </td><td> Lonza </td><td> 50101 </td></tr><tr><td> भेड़ सीरम </td><td> सिग्मा </td><td> S2263-500ml </td></tr><tr><td> Stericup फिल्टर यूनिट, 0.22μm, polyethersulfone, 500ml </td><td> Millipore </td><td> SCGPU05RE </td></tr><tr><td> सोडियम azide दानेदार, </td><td> फिशर साइंटिफिक </td><td> S227I - 100 </td></tr><tr><td> सोडियम क्लोराइड </td><td> फिशर साइंटिफिक </td><td> BP358 - 212 </td></tr><tr><td> सोडियम dodecyl सल्फेट </td><td> फिशर साइंटिफिक </td><td> S529 - 500 </td></tr><tr><td> एसएससी, 20X समाधान </td><td> अनुसंधान उत्पाद इंटरनेशनल </td><td> S24022-4000.0 </td></tr><tr><td> सुपरस्क्रिप्ट III के संश्लेषण पहली भूग्रस्त प्रणाली </td><td> Invitrogen </td><td> 18080-051 </td></tr><tr><td> T7 शाही सेना पोलीमरेज़ </td><td> Roche एप्लाइड साइंस </td><td> 10881767001 </td></tr><tr><td> Taq पोलीमरेज़ डीएनए </td><td> Qiagen </td><td> 201203 </td></tr><tr><td> Tris - एचसीएल </td><td> फिशर साइंटिफिक </td><td> BP153 - 1 </td></tr><tr><td> 20 बीच </td><td> फिशर साइंटिफिक </td><td> BP337 - 100 </td></tr><tr><td> डीलक्स नमूना स्नान के साथ सूक्ष्म तक्षणी हिल </td><td> Leica माइक्रोसिस्टम्स </td><td> VT1000A </td></tr><tr><td> खमीर tRNA </td><td> Roche एप्लाइड साइंस </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

यहाँ, हम एक कुशल उच्च throughput का वर्णन<em&gt; बगल में</em&gt; भ्रूण माउस प्रोस्टेट ऊतक वर्गों के विकास में mRNA अभिव्यक्ति के पैटर्न को दृश्यमान करने के लिए संकरण विधि (ISH). विधि अन्य माउस ऊतकों में या अन्य प्रजातियों से ऊतकों में mRNA अभिव्यक्ति पैटर्न कल्पना आसानी से अनुकूलित कर सकते हैं.

एक उच्च Throughput बगल में संकरण विधि भ्रूण लोअर मूत्रजननांगी माउस पथ में mRNA अभिव्यक्ति पैटर्न विशेषताएँ

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

एक उच्च throughput के लिए विश्व स्तर पर एस cerevisiae में organelle आकारिकी अध्ययन विधि

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish पूरा पर्वत उच्च संकल्प डबल प्रतिदीप्त स्वस्थानी में संकरण

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

मानचित्रण Ribonucleoproteins के लिए एक रैपिड मानव पूर्व mRNA पर उच्च throughput विधि (RNPs)

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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जीन Pulser MXcell electroporation प्रणाली का उपयोग करने के लिए उच्च क्षमता के साथ प्राथमिक कोशिकाओं transfect

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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हाय-C: विधि अध्ययन जीनोम के तीन आयामी वास्तुकला.

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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पूरे माउंट<em&gt; स्वस्थानी में</em&gt; E8.5 E11.5 माउस भ्रूण संकरण

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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रेडियोधर्मी बगल में ऊतक में विविध जीन एक्सप्रेशन पैटर्न का पता लगाने के लिए संकरण

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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उच्च throughput स्वचालित का उपयोग गुणसूत्रों की शारीरिक मानचित्रण<em&gt; बगल में</em&gt; संकरण

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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प्रतिदीप्ति का प्रयोग mRNA अणुओं के दृश्य और विश्लेषण<em&gt; स्वस्थानी</emमें&gt; संकरण<em&gt; 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 ...