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

The authors would like to thank Dr. Lan Yi, Cancer Institute of New Jersey, for technical assistance in preparing tissue baskets. This work was funded by National Institutes of Health Grants DK083425 and DK070219.

1. Synthesis of a Digoxigenin-11-UTP-Labeled Riboprobe from a PCR-Generated Template
<ol>
<li>To synthesize a gene-specific riboprobe, use Entrez Gene (http://www.ncbi.nlm.nih.gov/sites/entrez) to obtain the gene cDNA reference sequence (RefSeq). Use the Primer3 program (http://frodo.wi.mit.edu/primer3/)5 to design gene-specific PCR primers against the 3&#x2019;-region of the cDNA sequence. Recommended parameters for PCR primer selection are described elsewhere (http://www.gudmap.org/Research/Protoc...

<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>

Using the method described here, it is possible to detect mRNAs in all of the major cell types and tissue compartments of the fetal male and female mouse LUTs including the mesenchymal pads, urothelium, smooth muscle, prostatic buds, ejaculatory duct, and vagina. The 50&mu;m sections used in this protocol have the advantage of being thick enough to resolve tissue architecture (such as blood vessels) but are thin enough to avoid probe trapping, which is a methodological problem commonly encountered during whole-mount ISH....

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>Anti-Digoxigenin antibody, Fab fragments</td>
 <td>Roche Applied Science</td>
 <td>11214667001</td>
 </tr>
 <tr>
 	<td>Blocking reagent</td>
 <td>Roche Applied Science</td>
 <td>11096176001</td>
 </tr>

 <tr>
 	<td>BM Purple AP substrate, precipitating</td>
 <td>Roche Applied Science</td>
 <td>11442074001</td>
 </tr>
 <tr>
 	<td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 </tr>
 <tr>
 	<td>Cell culture plate, 24 well</td>
 <td>Corning</td>
 <td>3524</td>
 </tr>
 <tr>
 	<td>Digoxigenin 11-UTP</td>
 <td>Roche Applied Science</td>
 <td>1277073910</td>
 </tr>
 <tr>
 	<td>dNTPs</td>
 <td>Roche Applied Science</td>
 <td>11969064001</td>
 </tr>
 <tr>
 	<td>Double-edged razor blade</td>
 <td>Wilkinson Sword</td>
 <td>Classic Model</td>
 </tr>
 <tr>
 	<td>Eliminase RNase remover</td>
 <td>Decon Laboratories</td>
 <td>1102</td>
 </tr>
 <tr>
 	<td>Formamide</td>
 <td>Sigma</td>
 <td>F5786-1L</td>
 </tr>
 <tr>
 	<td>Gel extraction kit</td>
 <td>Qiagen</td>
 <td>28704</td>
 </tr>
 <tr>
 	<td>Glutaraldehyde, 25% solution in H2O</td>
 <td>Sigma</td>
 <td>G6257-100ML</td>
 </tr>
 <tr>
 	<td>Heparin, sodium salt</td>
 <td>Sigma</td>
 <td>H3393</td>
 </tr>
 <tr>
 	<td>Hydrogen peroxide, 30% solution in H2O</td>
 <td>Fisher Scientific</td>
 <td>BP2633-500</td>
 </tr>
 <tr>
 	<td>Levamisole</td>
 <td>Sigma</td>
 <td>L9756</td>
 </tr>
 <tr>
 	<td>Loctite 404 quick set instant adhesive</td>
 <td>Henkel Corp.</td>
 <td>46551</td>
 </tr>
 <tr>
 	<td>Magnesium chloride</td>
 <td>Fisher Scientific</td>
 <td>M33-500</td>
 </tr>
 <tr>
 	<td>Maleic acid</td>
 <td>Sigma</td>
 <td>M0375-500G</td>
 </tr>
 <tr>
 	<td>Microcentrifuge tubes, 1.5mL</td>
 <td>Biologix Research Company</td>
 <td>BP337-100</td>
 </tr>
 <tr>
 	<td>Millicell culture plate insert</td>
 <td>Millipore</td>
 <td>PICM01250</td>
 </tr>
 <tr>
 	<td>Molecular grinding resin</td>
 <td>G-Biosciences</td>
 <td>786-138PR</td>
 </tr>
 <tr>
 	<td>Paraformaldehyde, 4% solution in phosphate buffered saline</td>
 <td>Affymetrix</td>
 <td>19943</td>
 </tr>
 <tr>
 	<td>Phosphate buffered saline, without Ca &amp; Mg</td>
 <td>MP Biomedicals</td>
 <td>ICN1760420</td>
 </tr>
 <tr>
 	<td>Polyester mesh, 33 micron, 12&#x201D; x 24&#x201D;</td>
 <td>Small Parts Inc</td>
 <td>CMY-0033-D</td>
 </tr>
 <tr>
 	<td>Proteinase K solution, 20mg/ml</td>
 <td>Amresco</td>
 <td>E195-5ML</td>
 </tr>
 <tr>
 	<td>QIAshredder Columns</td>
 <td>Qiagen</td>
 <td>79654</td>
 </tr>
 <tr>
 	<td>Q solution</td>
 <td>Qiagen</td>
 <td>Provided with Taq DNA polymerase</td>
 </tr>
 <tr>
 	<td>RNase</td>
 <td>Sigma</td>
 <td>R6513</td>
 </tr>
 <tr>
 	<td>RNase inhibitor</td>
 <td>Roche Applied Science</td>
 <td>03335399001</td>
 </tr>
 <tr>
 	<td>RNeasy mini kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 	<td>RNEASY mini kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 	<td>RQ1 RNase-free DNase</td>
 <td>Promega</td>
 <td>M6101</td>
 </tr>
 <tr>
 	<td>SeaPlaque low-melt agarose</td>
 <td>Lonza</td>
 <td>50101</td>
 </tr>
 <tr>
 	<td>Sheep serum</td>
 <td>Sigma</td>
 <td>S2263-500mL</td>
 </tr>
 <tr>
 	<td>Stericup filter unit, 0.22&mu;m, polyethersulfone, 500mL</td>
 <td>Millipore</td>
 <td>SCGPU05RE</td>
 </tr>
 <tr>
 	<td>Sodium azide, granular</td>
 <td>Fisher Scientific</td>
 <td>S227I-100</td>
 </tr>
 <tr>
 	<td>Sodium chloride</td>
 <td>Fisher Scientific</td>
 <td>BP358-212</td>
 </tr>
 <tr>
 	<td>Sodium dodecyl sulfate</td>
 <td>Fisher Scientific</td>
 <td>S529-500</td>
 </tr>
 <tr>
 	<td>SSC, 20X solution</td>
 <td>Research Products International</td>
 <td>S24022-4000.0</td>
 </tr>
 <tr>
 	<td>SuperScript III first-strand synthesis system</td>
 <td>Invitrogen</td>
 <td>18080-051</td>
 </tr>
 <tr>
 	<td>T7 RNA polymerase</td>
 <td>Roche Applied Science</td>
 <td>10881767001</td>
 </tr>
 <tr>
 	<td>Taq DNA polymerase</td>
 <td>Qiagen</td>
 <td>201203</td>
 </tr>
 <tr>
 	<td>Tris-HCl</td>
 <td>Fisher Scientific</td>
 <td>BP153-1</td>
 </tr> 
 <tr>
 	<td>Tween 20</td>
 <td>Fisher Scientific</td>
 <td>BP337-100</td>
 </tr> 
 <tr>
 	<td>Vibrating microtome with deluxe specimen bath</td>
 <td>Leica Microsystems</td>
 <td>VT1000A</td>
 </tr> 
 <tr>
 	<td>Yeast tRNA</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

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

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 ...

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