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/Protocols/Vezina/Riboprobe_Syn.html).</li>
<li>Use the MegaBLAST Program (http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=10090)6 to assess specificity of the selected DNA sequence. The DNA sequence is considered specific when, using an EXPECT threshold of 0.01, it does not align with other sequences in the RefSeq database.</li>
<li>PCR amplify the riboprobe template. PCR reaction components and thermocycling conditions should be optimized for each primer set. A typical 50 &mu;l reaction contains: 1X buffer, 2mM MgCl2, 0.2mM dNTPs, 1X Q solution, 1&mu;g cDNA, 2.5U Taq DNA polymerase, 0.25&mu;m primers, and nuclease-free H2O. A typical thermocycling protocol includes an initial denaturation at 94&deg;C for 2min followed by 40 cycles of 94&deg;C for 30sec, 57&deg;C for 30sec, 72&deg;C for 1min and a final extension at 72&deg;C for 10min. The cDNA used in the PCR reactions is synthesized from mouse urogenital mRNA.</li>
<li>Separate the PCR product by agarose gel electrophoresis, purify using the Gel Extraction Kit, and quantify the purified products by spectrophotometry. The expected yield is 1.2 - 3.6&mu;g.</li> 
<li>Transcribe the PCR product into a labeled riboprobe. The transcription reaction (40 &mu;l) contains: 400ng purified PCR product, 1X nucleotide labeling mix containing digoxigenin 11-UTP, 1X transcription buffer, 5U RNase inhibitor, 80U T7 RNA polymerase, and nuclease-free H2O. Incubate 3-4hr at 37&deg;C and agitate samples every 30min.</li>
<li>Use the Qiagen RNEASY Mini kit to purify riboprobes based on instructions for RNA cleanup with on-column DNase digestion. Quantitate riboprobes by spectrophotometry. The expected yield is 4-20 &mu;g. Assess probe quality by separating an aliquot by electrophoresis on a 1.5% non-denaturing agarose gel. High quality probes migrate as distinct bands with minimal smearing.</li>
<li>To ensure the riboprobe specifically recognizes its target, include a positive control tissue for the first ISH experiment. The riboprobe&#x2019;s target mRNA pattern should be known in this positive control tissue.</li>
</ol>

2. Preparation of Vibrating Microtome Blade (Based on Previously Described Protocol)7
<ol>
<li>Prepare a 4% low-melt agarose solution in phosphate buffered saline (PBS). Microwave solution to dissolve agarose and maintain solution at 62&deg;C.</li> 
<li>Prepare a polystyrene ring mold by removing the membrane from a 12mm diameter Millicell culture plate well insert and retain polystyrene ring to use as an agarose mold. Soak the rings overnight in RNase inhibitor solution prior to each use.</li>
<li>To ensure a smooth cutting surface, remove rust inhibitor and other additives from the surface of the Wilkinson blade by rinsing with the following solvents, at 100% concentration: petroleum ether, xylene, chloroform, methanol, and MilliQ water. Separate the double blade lengthwise into two single blades.</li>
<li>Adhere the Wilkinson blades to a microtome blade with Loctite adhesive. The microtome blade is not used for cutting; it adds rigidity to the Wilkinson blade. The microtome blade should be cut to the length of the Wilkinson blade using metal shears and, once adhered, should be offset by 3 &#x2013; 4mm from the cutting edge of the Wilkinson blade. </li>
</ol>

3. Dissection, Storage, and Preparation of Urogenital Tissues for Sectioning
<ol>
<li>Prepare PBSTw solution (PBS containing 0.1% Tween&trade; 20 and 0.2mM sodium azide, filtered through the 0.22&mu;m Stericup&reg; filter unit). Solution can be prepared in advance and stored at 25&deg; C.</li>
<li>Incubate a freshly dissected mouse LUT (bladder, pelvic urethra, and associated Wolffian and M&#x03CB;llerian duct-derived structures) overnight at 4&deg;C in PBS containing 4% paraformaldehyde fixative.</li> 
<li>Dehydrate tissues by washing for 10min at 25&deg;C in a series of graded methanol/PBSTw (1:3, 1:1, 3:1 v/v) solutions. Store samples at &#x2013;20&deg;C in 100% methanol at least overnight. Archived tissues may be kept at least 2yr.</li> 
<li>Prepare tissues for sectioning by rehydrating archived tissues. Wash for 10min at 25&deg;C in a series of graded methanol/PBSTw (3:1, 1:1, 1:3 v/v) solutions.</li>
<li>Dissect and discard approximately two-thirds of the bladder, leaving most of the trigone region attached to the urethra.</li>
</ol>


4. Embedding Urogenital Tissue in Agarose
<ol>
<li>Place the polystyrene ring mold, flat surface down, on a 25&deg;C plain glass microscope slide.</li>
<li>Fill the ring mold with 62&deg;C agarose solution and cool for about 2min.</li>
<li>Remove LUT tissue from PBSTw and blot dry on an absorbent wipe.</li> 
<li>Transfer the tissue into agarose solution.</li>
<li>Use forceps to orient the LUT tissue in agarose so that it is suspended halfway between the top and bottom of the ring mold and incubate the tissue at 4&deg;C until the agarose has solidified.</li>
<li>If the tissue sinks completely during the process of agarose solidification, it can be excised from the agarose and re-embedded. Adjust the cooling time of the agarose as needed during the re-embedding process.</li> 
</ol>

5. Sectioning Urogenital Tissue With a Vibrating Microtome (Based on Previously Described Protocol)7
<ol>
<li>Mount the reinforced Wilkinson blade in the vibrating microtome and set the blade angle to 35&deg;. Fill deluxe specimen bath with PBS and pack wet ice around the specimen bath.</li>
<li>Remove the solidified agarose plug from the ring mold and blot the bottom surface with an absorbent wipe. Verify that the tissue is oriented correctly. Tissues orientation can be adjusted by using a razor blade to bevel the flat edge of the agarose plug.</li>
<li>Adhere the agarose plug onto a vibrating microtome specimen mounting disk with Loctite adhesive as shown in Fig. 1A.</li>
<li>Insert the specimen mounting disk into the vibratome.</li> 
<li>Adjust the microtome section thickness to 50&mu;m, the speed to 2, and the blade amplitude to 4 and begin cutting tissue sections.</li>
<li>Use blunt forceps to transfer each tissue section (Fig. 1B) to a 24-well culture plate well that contains ice-cold 0.5mL PBSTw.</li>
<li>To prepare samples for in situ hybridization, excise most of the agarose around each tissue section (the remaining agarose will melt during the ISH procedure) and remove all associated debris. Store sections up to 48hr at 4&deg;C in PBSTw.</li>
</ol>
6. Sample Basket Preparation For In Situ Hybridization
<ol>
<li>Cut the bottom of a microcentrifuge tube at the 100&mu;L mark.</li>
<li>Heat the cut edge of the tube in a flame until the plastic is softened, then press the microcentrifuge tube firmly onto the center of a 0.5in polyester mesh square.</li> 
<li>Trim excess mesh and use a heated 18 gauge needle to pierce two holes into each tube lid to complete basket preparation (Fig. 1C).</li> 
<li>Remove the lid of a 24 well plate and drill a 12mm hole centered over each well. Use the lid to transfer sample baskets between washes of the ISH protocol (Fig. 1D).</li>
</ol>
7. Embryo Powder Preparation For In Situ Hybridization (Based on Previously Described Protocol)8
<ol>
<li>Collect mouse embryo tissue from mice that are the same stage as the tissue sections that are being assessed and store at &#x2013;80&deg;C. Place frozen tissue into a ceramic mortar, submerge tissue in liquid nitrogen, and use a pestle to grind tissue into a fine powder.</li> 
<li>Combine embryo powder with 4 volumes of acetone and homogenize with several strokes of a dounce homogenizer.</li>
<li>Transfer homogenate to a 15mL glass screw-top vial and extract overnight at 4&deg;C.</li>
<li>Pellet embryo powder by centrifugation at 5000rpm for 10min at 4&deg;C. Remove and discard the lipid-containing supernatant. Resuspend the tissue pellet in 4vol of fresh acetone and extract for 2hr at 4&deg;C.</li>
<li>Pellet the embryo powder by centrifugation at 5000rpm for 10min at 4&deg;C. Remove and discard the supernatant.</li>
<li>Air dry the pellet on a #2 Whatman filter paper. Crush pellet to yield a fine powder and store in a tightly sealed glass vial at 4&deg;C. The approximate yield is 50 mg powder per 1 g embryo wet weight.</li>
</ol>

8. In Situ Hybridization Day 1
<ol>
<li>Preheat prehybridization solution (50% formamide, 5x SSC, 1% Blocking reagent, 10&mu;g/mL yeast tRNA, 10&mu;g/mL heparin store at -20&deg;C) to 60.5&deg;C. This solution can be prepared in advance and stored at -20&deg;C.</li>
<li>Prepare a humidified hybridization chamber by filling a small plastic storage container with about 0.5 in of tap water. Cover container and preheat to 60.5&deg;C.</li> 
<li>Add 2mL PBSTw to the wells of a 24-well culture plate. Place sample baskets in the holes of the 24-well plate lid and transfer tissue sections into the baskets (up to 10 sections per basket have been used). </li>
<li>Incubate tissue sections for 30min at 25&deg;C in 6% H2O2. This and all subsequent incubations should be carried out with gentle agitation on an orbital shaker, unless otherwise indicated. All incubations and washes are conducted in 24-well plates and use a total solution volume of 2mL/well.</li>
<li>Wash tissue sections 4 x 5min at 25&deg;C in PBSTw.</li>
<li>Incubate tissue sections for 12min at 25&deg;C in PBSTw containing 5&mu;g/mL proteinase K.</li>
<li>Wash tissue sections 1 x 5min at 25&deg;C in PBSTw.</li>
<li>Post-fix tissue sections for 20min at 25&deg;C in PBS containing 4% paraformaldehyde and 0.2% glutaraldehyde.</li>
<li>Wash tissue sections 2 x 5min at 25&deg;C in PBSTw.</li>
<li>Add 2mL/well of the prewarmed prehybridization buffer and incubate tissue sections inside humidified hybridization chamber for at least 1hr at 60.5&deg;C.</li>
<li>Add 0.65&mu;g labeled riboprobe to the prehybridization buffer in each well and incubate tissue sections overnight in the humidified hybridization chamber at 60.5 &deg;C.</li>
</ol>
9. In Situ Hybridization Day 2
<ol>
<li>Prepare the following solutions for post-hybridization washing steps: Solution 1 (50% formamide, 5x SSC, 1% SDS), Solution 2 (10mM Tris-HCL pH 7.5, 0.5M NaCl, 0.1% Tween&trade; 20, 0.2mM sodium azide, 0.22&mu;m filtered), and Solution 3 (2x SSC, 50% formamide). These solutions can be prepared in advance. Solutions 1 and 3 are stored at -20&deg;C and Solution 2 is stored at 25&deg;C. The storage life of the solutions is at least 3 months.</li>
<li>Wash tissue sections 3 X 30min at 60.5&deg;C with pre-warmed Solution 1. Use the humidified chamber during washes.</li>
<li>Wash tissue sections 1 X 10min at 60.5&deg;C with pre-warmed Solution 1/Solution 2 (1:1 v/v) solution. Use the humidified chamber during the wash.</li>
<li>Wash tissue sections 4 X 10min at 25&deg;C with Solution 2.</li> 
<li>Incubate tissues sections for 15min at 37&deg;C in Solution 2 containing 0.25&mu;g/mL RNase.</li>
<li>Wash tissue sections 1 X 10min at 25&deg;C with Solution 2 (without RNase).</li> 
<li>Wash tissue sections 1 X 10min at 25&deg;C with Solution 3, followed by 2 X1hr washes at 60.5&deg;C with Solution 3. Use the humidified chamber during the 60.5&deg;C washes.</li>
<li>Prepare the following solutions for immunohistochemical detection of the DIG-labeled riboprobes: Tissue Blocking Buffer (TB, 1X TBS, 10% sheep serum, 1% blocking reagent, 1% BSA, 0.1% Tween&trade; 20, 0.22&mu;m filtered), Antibody Dilution Buffer (AD, 1xTBS, 5% sheep serum, 1% blocking reagent, 1% BSA, 0.1% Tween&trade; 20, 0.2mM sodium azide, 0.22&trade;m filtered) Antibody Absorption Buffer (AA, 1xTBS, 5% sheep serum, 1% blocking reagent, 1% BSA, 6mg/mL embryo powder), and TBSTw (1xTBS, 0.1% Tween&trade; 20, 0.2mM sodium azide, 0.22&mu;m filtered). These solutions can be prepared in advance. TBSTw is stored at 25&deg;C, all other solutions are stored at -20&deg;C.</li>
<li>Wash 3 X 10 min at 25&deg;C with TBSTw.</li>
<li>Incubate the tissue sections at least 2hr at 25&deg;C in TB buffer.</li>
<li>While tissues are incubating in TB buffer, add 1.1&mu;L anti-DIG antibody per 200&mu;L AA buffer for each well. Incubate AA buffer + antibody at least 2hr at 4&deg;C, then centrifuge at 10,000rpm for 1min. Remove the AA buffer supernatant and add it to 2mL of AD buffer.</li>
<li>Remove tissue sections from TB buffer and incubate them overnight in a humidified chamber at 4&deg;C in AD buffer containing antibody.</li>
</ol>
10. In Situ Hybridization Day 3
<ol>
<li>Prepare color development solution NTMT (100mM Tris-HCL pH 9.5, 100mM NaCl, 50mM MgCl2, 0.2mM sodium azide, 0.22&mu;m filtered). This solution can be prepared in advance and stored at 25&deg;C. Immediately prior to use, add 2mM levamisole and 0.1% Tween&trade; 20.</li> 
<li>Remove the antibody solution (AD buffer + antibody) from the wells and store solution at 4&deg;C. It can be reused up to two additional times.</li>
<li>Wash the tissues 8 X 10 min at 25&deg;C with TBSTw containing 2mM levamisole.</li>
<li>Carefully transfer tissues from the baskets to a Petri dish containing TBSTw. Use forceps to remove visible debris. Transfer the tissues into clean microcentrifuge tubes.</li>
<li>Wash tissues 1 X 10 min at 25&deg;C with 1mL NTMT.</li>
<li>Remove the NTMT and add 1mL/tube of a mixture containing 50% NTMT (containing 2mM levamisole) and 50% BM Purple, place tubes in a light protected box and incubate at 25&deg;C. Color development time ranges from several hours to several days. If color is slow to develop, 100% BM Purple can be used.</li> 
<li>Monitor color development and change NTMT/BM Purple solution if it accumulates precipitated crystals or if it undergoes color change from yellow to purple. After full color development (4-250 hours), wash tissues 2 X 5min at 25&deg;C with 1mL/tube of NTMT containing 2mM levamisole.</li>
<li>Incubate tissues overnight at 4&deg;C in 1mL/tube of PBS containing 4% paraformaldehyde post-fixative.</li>
<li>To bleach the tissues, incubate tissues for 30min at 25&deg;C in 1mL/tube of PBSTw containing 3% H2O2. Then wash the tissues 1 X 10min at 25&deg;C in 1mL/tube of PBSTw and store at 4&deg;C in 1mL/tube of PBS containing 4% paraformaldehyde post-fixative.</li>
<li>Tissue sections are mounted on glass slides, coverslipped, and imaged with a compound microscope. </li>
</ol>

11. Representative Results: 
The spatial orientation of LUT tissue in agarose determines the plane of tissue sections. For sagittal sections, at least two-thirds of the bladder is excised and the remaining LUT tissue is embedded in agar such that the urethral midline is parallel to the flat surface of the agarose plug (Figure 1A). Minor adjustments in the tissue plane can be made by beveling the flat edge of the agarose plug. A representative sagittal section from a 17.5dpc male LUT tissue that is oriented in this plane is shown in Figure 1B. 
Sample baskets protect the delicate tissue sections from loss and from accumulating dust and particulate matter during the multi-day ISH procedure. Sample baskets are prepared by melting polyester mesh to the cut end of a 1.5mL microfuge tube (Figure 1C). A small hole is pierced into the lid of each sample basket to facilitate solution flow into and out of the baskets. Sample baskets are suspended in ISH solutions by placing them in 12mm holes drilled into 24-well plate lids (Figure 1D). The modified plate lids support baskets when they are transferred between 24-well plates during solution changes.
It is challenging to limit non-specific background staining during the long incubation periods required for detection of low abundance mRNAs. The addition of 0.2mM sodium azide to sample buffers and their subsequent filtration through 0.22&mu;m filters appeared to limit background staining (Figure 2). Using the method described here, there does not appear to be visible differences in background staining when samples are incubated in color development solution for a prolonged time periods (Figure 3). 
<img src="/files/ftp_upload/2912/2912fig1.jpg" alt="Figure 1"/> Figure 1. Preparation of a mouse lower urogenital (LUT) tract tissue section and microcentrifuge tube a basket for ISH. A LUT containing part of the bladder, pelvic urethra and associated Wolffian and M&#x03CB;llerian duct-derived structure) is embedded in a cylindrical plug of 4% low-melt agarose. (A) The plug is glued to a specimen mounting disk and (B) cut into 50&mu;m sections with a vibrating microtome. (C) An LUT section is transferred into a microcentrifuge tube basket that is prepared by piercing a hole into the tube lid and fusing polyester mesh to the cut bottom end of the tube. (D) The microcentrifuge tube is inserted into 12mm holes drilled into a 24-well plate lid so that tissue sections are suspended in buffer solution during the ISH protocol. Arrowheads indicate the LUT tissue in the agarose plug.
<img src="/files/ftp_upload/2912/2912fig2.jpg" alt="Figure 2"/> Figure 2. Incorporation of 0.2mM sodium azide into 0.22&mu;m filtered solutions improves tissue quality and reduces background staining. 17.5dpc male mouse lower urogenital tracts (LUTs) were sectioned in a sagittal plane to a thickness of 50&mu;m. Tissue sections were stained by ISH using a probe directed against twist homolog 1. Buffers used for ISH were either (A) 0.22&mu;m filtered and supplemented with 0.2mM sodium azide (NaAz) or (B) unfiltered and not supplemented with NaAz. Arrowheads indicate background staining. Images were captured at the same magnification. Results are representative staining patterns for n = 3 litter-independent mice.
<img src="/files/ftp_upload/2912/2912fig3.jpg" alt="Figure 3"/> Figure 3. Background staining intensity does not appear to increase with prolonged color development. 17.5dpc male mouse lower urogenital tracts (LUTs) were sectioned in a sagittal plane to a thickness of 50&mu;m. Sections were stained by ISH by incubating them in chromagen staining solution for (A) 9.5h using a probe that recognizes the high abundance transcript estrogen-related receptor gamma (Esrrg), (B) for 43.5h using a probe that recognizes the medium abundance transcript bromodomain adjacent to zinc finger domain, 2A (Baz2a), or (C) for 236h using a probe that recognizes the low abundance transcript wingless-type MMTV integration site family, member 10a (Wnt10a). Images were captured at the same magnification. Results are representative staining patterns for n = 3 litter-independent mice.

<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>

No conflicts of interest declared.

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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JoVE Journal

Biology

12.9K Views.  University of Wisconsin-Madison. 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.

Video: 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

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

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

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

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

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



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

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

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.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

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 is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

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

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

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

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. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

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

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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

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 over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

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

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

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

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

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

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

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

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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