Name: radioactive in situ hybridization for detecting diverse gene expression patterns in tissue
Uploaded: 2012-04-27T13:00:00
Duration: 17 min 38 s
Description: Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

The authors would like to thank all Jarvis lab members who improved the protocol over the years.

<ol>
	<li>Clayton, D. F., Huecas, M. E., Sinclair-Thompson, E. Y., Nastiuk, K. L., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probes+for+rare+mRNAs+reveal+distributed+cell+subsets+in+canary+brain.">Probes for rare mRNAs reveal distributed cell subsets in canary brain.</a> Neuron. 1, 249-261 (1988).</li><li>Wada, K., Sakaguchi, H., Jarvis, E. D., Hagiwara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+glutamate+receptors+in+avian+neural+pathways+for+learned+vocalization.">Differential expression of glutamate receptors in avian neural pathways for learned vocalization.</a> J. Comp. Neurol. 476, 44-64 (2004).</li><li>Haesler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP2+expression+in+avian+vocal+learners+and+non-learners.">FoxP2 expression in avian vocal learners and non-learners.</a> J. Neurosci. 24, 3164-3175 (2004).</li><li>Jarvis, E. D., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor-driven+gene+expression.">Motor-driven gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 4097-4102 (1997).</li><li>Holzenberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+insulin-like+growth+factor+II+in+the+songbird+brain.">Selective expression of insulin-like growth factor II in the songbird brain.</a> J. Neurosci. 17, 6974-6987 (1997).</li><li>Mahmood, R., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+hybridization+of+radioactive+riboprobes+to+RNA+in+tissue+sections.">In-situ hybridization of radioactive riboprobes to RNA in tissue sections.</a> Methods Mol. Biol. 461, 675-686 (2008).</li><li>Wilkinson, D. G., Nieto, M. A. <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 Enzymol. 225, 361-373 (1993).</li><li>Acloque, H., Wilkinson, D. G., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+analysis+of+chick+embryos+in+whole-mount+and+tissue+sections.">In situ hybridization analysis of chick embryos in whole-mount and tissue sections.</a> Methods Cell Biol. 87, 169-185 (2008).</li><li>Moorman, A. F., Houweling, A. C., de Boer, P. A., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+nonradioactive+detection+of+mRNA+in+tissue+sections:+novel+application+of+the+whole-mount+in+situ+hybridization+protocol.">Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.</a> J. Histochem. Cytochem. 49, 1-8 (2001).</li><li>Horita, H., Wada, K., Rivas, M. V., Hara, E., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dusp1+immediate+early+gene+is+regulated+by+natural+stimuli+predominantly+in+sensory+input+neurons.">The dusp1 immediate early gene is regulated by natural stimuli predominantly in sensory input neurons.</a> J. Comp. Neurol. 518, 2873-2901 (2010).</li><li>Braissant, O., Wahli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+in+situ+hybridization+protocol+using+non-radioactively+labelled+probes+to+detect+abundant+and+rare+mRNAs+on+tissue+sections.">A simplified in situ hybridization protocol using non-radioactively labelled probes to detect abundant and rare mRNAs on tissue sections.</a> Biochemica. 1, 10-16 (1998).</li><li>Kubikova, L., Wada, K., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+receptors+in+a+songbird+brain.">Dopamine receptors in a songbird brain.</a> J. Comp. Neurol. 518, 741-769 (2010).</li><li>Wada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+neuroethological+approach+for+identifying+and+characterizing+a+cascade+of+behaviorally+regulated+genes.">A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 15212-15217 (2006).</li><li>Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+brains+and+a+new+understanding+of+vertebrate+brain+evolution.">Avian brains and a new understanding of vertebrate brain evolution.</a> Nat. Rev. Neurosci. 6, 151-159 (2005).</li><li>Hoke, K. L., Ryan, M. J., Wilczynski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+shift+functional+connectivity+in+the+hypothalamus.">Social cues shift functional connectivity in the hypothalamus.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 10712-10717 (2005).</li><li>Burmeister, S. S., Jarvis, E. D., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+behavioral+and+genomic+responses+to+social+opportunity.">Rapid behavioral and genomic responses to social opportunity.</a> PLoS. Biol. 3, e363 (2005).</li><li>Jarvis, E. D., Schwabl, H., Ribeiro, S., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+gene+regulation+by+territorial+singing+behavior+in+freely+ranging+songbirds.">Brain gene regulation by territorial singing behavior in freely ranging songbirds.</a> Neuroreport. 8, 2073-2077 (1997).</li><li>Jarvis, E. D., Scharff, C., Grossman, M. R., Ramos, J. A., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+whom+the+bird+sings:+context-dependent+gene+expression.">For whom the bird sings: context-dependent gene expression.</a> Neuron. 21, 775-788 (1998).</li><li>Feenders, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mapping+of+movement-associated+areas+in+the+avian+brain:+a+motor+theory+for+vocal+learning+origin.">Molecular mapping of movement-associated areas in the avian brain: a motor theory for vocal learning origin.</a> PLoS One. 3, e1768 (2008).</li><li>Chen, C. C., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+two+gonadotropin-releasing+hormone+receptor+types+in+a+cichlid+fish+suggest+functional+specialization.">Distributions of two gonadotropin-releasing hormone receptor types in a cichlid fish suggest functional specialization.</a> J. Comp. Neurol. 495, 314-323 (2006).</li></ol>

We have nothing to disclose.

Radioactive in situ hybridization of mRNA expression is widely used for multiple purposes, including for studying regional tissue organization, cell types, and brain functional activity2-5,10,12-14. The later use is on genes whose mRNA expression in the brain is dependent on increased neural activity, often called activity-dependent genes or immediate early genes. With these uses, our method has been applied across multiple species, including in birds, mammals (e.g. human), fish, and amphibians; in multiple tissues, including brain, skin, and muscle; and multiple ages, including hatchlings/neonates, juveniles, adults, and here in whole embryo sections2,3,5,15-17. The special features of our protocol include: (1) It produces a balance between anatomical specificity and quantitative specificity. To quantify the gene expression on the x-ray film, we take digital pictures of the images (ex: Fig. 1A and 1B), use the Photoshop (Adobe) histogram function to measure the pixel density in the regions of interest and subtract the background levels on the film outside of the tissue but still on the glass slide2,4. To quantify expression at the cellular level, we take images of silver grains over cells under high magnification (40-100X; Fig 4). We then use the threshold and measure functions of Image J by Wayne Rasband at NIH to count the number of silver grains in the image, subtract out the background count in a similar area without cells on the glass slide, divide by the number of cells, to obtain a values of expression per cell.4,18 (2) It can be relatively high throughput, allowing processing of 100-200 slides simultaneously, due to the tight coverslip seal created by the mineral oil bath. Standard in-situ hybridization methods take longer to seal the slides with parafilm, nail polish, and other means, where slides take up a lot of space; (3) It is highly sensitive for low abundance transcripts due to Dextran sulfate and Denhardt's solution in the hybridization buffer2,13; (4) The imaging approach in darkfield yields high contrast images due to taking pictures under darkfield illumination on a dissecting microscope4,5. Additionally, It allows sensitive detection of small changes in gene expression, such as in activity-dependent gene expression to identify specific brain regions activated during perception and production of specific behaviors19. The limitations relative to non-radioactive protocols are that the latter are clearer in the cellular resolution and location of the mRNA within the cell, and working with emulsion is very sensitive to manipulation and light. It is possible to combine our method with other methods, such as non-radioactive in situ hybridization to label mRNA expression of more than one gene in the same tissue10,17,20. It can be combined with immunocytochemistry to label both RNA and protein expression on the same sample in order to co-localize the mRNA with certain cell types10. The protocol modifications necessary for such double labeling experiments are described in the cited references.
In summary, our approach facilitates understanding of the timing and cellular location of gene expression in order to understanding region organization, tissue functional activity, and gene function.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Acetic anhydride</td>
 <td>VWR</td>
 <td>MK242002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>VWR</td>
 <td>BDH1109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cresyl violet acetate </td>
 <td>Sigma</td>
 <td>C5042</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Thermo scientific</td>
 <td>Microm HM550</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deionized formamide</td>
 <td>Sigma</td>
 <td>F9037</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Promega</td>
 <td>P1171</td>
 <td>100mM</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma</td>
 <td>ED</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Embedding mold</td>
 <td>VWR</td>
 <td>15160-215</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fisher brand Superfrost plus slide </td>
 <td>Fisher Scientific</td>
 <td>22-034-979</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>VWR</td>
 <td>BDH0506-4LP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formamide</td>
 <td>Sigma</td>
 <td>F7508</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GENECLEAN kit</td>
 <td>Q-Bio gene </td>
 <td>1001-200 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak BioMax MR film</td>
 <td>Sigma</td>
 <td>Z350370</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak NTB Emulsion</td>
 <td>Carestream Health</td>
 <td>8895666</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak Professional developer D19</td>
 <td>Kodak</td>
 <td>1462593</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak professional Fixer</td>
 <td>Kodak</td>
 <td>1971746</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&#946;-mercaptoethanol </td>
 <td>Calbiochem</td>
 <td>444203</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>VWR</td>
 <td>IC15169491</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>VWR</td>
 <td>SX0600-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Sigma</td>
 <td>76240</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly A</td>
 <td>Invitrogen</td>
 <td>POLYA.GF</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>rATP</td>
 <td>Promega</td>
 <td>P1132</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>rCTP</td>
 <td>Promega</td>
 <td>P1142</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>rGTP</td>
 <td>Promega</td>
 <td>P1152</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>RNasin</td>
 <td>Promega</td>
 <td>N2111</td>
 <td>40Units/&mu;l </td>
 </tr>
 <tr>
 <td>S35 UTP</td>
 <td>PerkinElmer</td>
 <td>NEG039C001MC </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Safety-Solve solution </td>
 <td>Safety Solve Research Products International </td>
 <td>111177 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Acetate</td>
 <td>Sigma</td>
 <td>S7899</td>
 <td>3M</td>
 </tr>
 <tr>
 <td>Sodium phosphate dibasic</td>
 <td>Sigma</td>
 <td>S3264</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium phosphate monobasic</td>
 <td>Sigma</td>
 <td>S3139</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SP6 RNA polymerase</td>
 <td>Promega</td>
 <td>P1085</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Staining metal rack</td>
 <td>Electron Microscopy Sciences</td>
 <td>70312-54 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T7 RNA polymerase</td>
 <td>Promega</td>
 <td>P2075</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue-Tek OCT</td>
 <td>Sakura </td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5x Transcription buffer</td>
 <td>Promega</td>
 <td>P1181</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triethanolamine</td>
 <td>VWR</td>
 <td>IC15216391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris-HCl (1 M, pH 8.0)</td>
 <td>VWR</td>
 <td>101449-446</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>tRNA</td>
 <td>Roche</td>
 <td>10109509001</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Solutions:
<ol>
 <li>Reagents for making of riboprobes: 1.5 &mu;l DNA template (0.2-0.3 &mu;g/&mu;l), 2 &mu;l of 5x optimized transcription buffer, 1 &mu;l of 100mM DTT (comes with Polymerase from Promega, mix well at room temperature), 0.3 &mu;l of RNasin (40 units/&mu;l), 1.5 &mu;l of AGC ribonucleotide mix solution, 3.5 &mu;l of S35 UTP, and 1 &mu;l RNA polymerase. Bring up to 10 &mu;l with nuclease-free water.</li>
 <li>AGC ribonucleotide mix solution: mix equal amounts of 10mM ATP, GTP and CTP together.</li>
 <li>Sodium acetate solution for EtOH precipitation to remove free S35 UTP: 40 &mu;l RNase and DNase free water, 5 &mu;l of 3M Sodium Acetate, and 125 &mu;l of 100% EtOH.</li>
 <li>Hybridization buffer (10ml stock): 5 ml of 100% deionized formamide, 600 &mu;l of 5M NaCl, 1M tris-HCl (pH = 8.0), 240 &mu;l of 0.5M EDTA (pH =8.0), 100 &mu;l of 100x Denhart's solution, 100 &mu;l of 1M DTT, 250 &mu;l of 20mg/ml tRNA, 125 &mu;l of 20 mg/ml poly A, and 1 g of Sodium Dextran Sulfate. Add DEPC-treated water to bring the total volume to 10 ml. Shake vigorously and then incubate at 55&deg;C until all sodium dextran sulfate is dissolved. Store the hybridization buffer in -20&deg;C, which is good for ~6 months. </li>
 <li>4% buffered paraformaldehyde solution: Add 40 g of paraformaldehyde in 760 ml distilled water in a flask designated for paraformaldehyde, heat to 50&deg;C on a hot plate while stirring. Add 320 &mu;l of 10N NaOH to help dissolve the paraformaldehyde. After dissolving (~10 min), add 100 ml of 10x PBS, and bring the volume up with distilled water until 1 liter. Stir and heat the solution until paraformaldehyde is dissolved. The pH should be 7.4.</li>
 <li>Acetylation buffer: 13.6 ml of triethanolamine plus 2.52 ml of acetic anhydride in 1 liter of distilled water.</li>
 <li>20x SSPE solution: 3M NaCl, 200 mM NaH2PO4-H2O, and 200 mM EDTA in distilled water. Adjust solution to pH 7.4 with 10N NaOH. </li>
 <li>10x PBS buffer: 80g of NaCl, 2.0g of KCl, 14.4g of Na2HPO4, and 2.4g of KH2PO4 in distilled water. Adjust solution to pH 7.0 and bring total volume to 1 liter. </li>
 <li>Second wash solution: 0.1% &#946;-mercaptoethanol and 50% formamide in 2x SSPE</li>
 <li>Cresyl violet acetate solution: 3% cresyl violet acetate in tap water (distilled water prevents good staining), dissolved overnight with stirring in a flask at room temperature. Filter with vacuum suction through a 1mm Whatman filter paper and Buchner funnel. </li> 
</ol>

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.

Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

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.

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. ...

radioactive-situ-hybridization-for-detecting-diverse-gene-expression

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

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 were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

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.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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.

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.

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

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...