Les auteurs tiennent à remercier tous les membres Jarvis laboratoire qui ont amélioré le protocole au fil des ans.

<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>

Nous n&#39;avons rien à communiquer.

Radioactifs hybridation in situ de l&#39;expression des ARNm est largement utilisé à des fins multiples, y compris pour l&#39;étude de l&#39;organisation des tissus régionaux, types de cellules, et l&#39;activité fonctionnelle du cerveau 2-5,10,12-14. L&#39;utilisation ultérieure est sur les gènes dont l&#39;expression d&#39;ARNm dans le cerveau dépend de l&#39;activité neuronale accrue, souvent appelés dépendants de l&#39;activité des gènes ou gènes précoces immédiats. Avec ces utilisations, notre méthode a été appliquée à travers de multiples espèces, y compris chez les oiseaux, les mammifères (par exemple l&#39;homme), les poissons et les amphibiens; dans de nombreux tissus, y compris le cerveau, la peau et le muscle, et les âges, y compris les nouveau-nés multiples ou les nouveau-nés, les jeunes , les adultes, et ici dans les sections d&#39;embryons entiers 2,3,5,15-17. Les caractéristiques particulières de notre protocole comprennent: (1) Elle produit un équilibre entre la spécificité anatomique et la spécificité quantitative. Afin de quantifier l&#39;expression des gènes sur le film x-ray, nous prenons des photos numériques des images (ex:. Fig 1A et 1B), utiliser le Photoshop fonction histogramme (Adobe) pour mesurer la densité de pixels dans les régions d&#39;intérêt et soustraire le bruit de fond sur le film extérieur du tissu mais encore sur la lame de verre 2,4. Pour quantifier l&#39;expression au niveau cellulaire, nous prenons des images de grains d&#39;argent sur ​​les cellules à fort grossissement (40-100X; Fig 4). Nous utilisons ensuite le seuil et les fonctions de mesure de J image par Wayne Rasband au NIH pour compter le nombre de grains d&#39;argent dans l&#39;image, il faut soustraire le nombre d&#39;arrière-plan dans un domaine similaire, sans cellules sur la lame de verre, il faut diviser par le nombre de cellules, pour obtenir un valeurs d&#39;expression par la cellule. 4,18 (2), il peut être débit relativement élevé, permettant le traitement de 100-200 lames simultanément, en raison de la lamelle d&#39;étanchéité étanche créée par le bain d&#39;huile minérale. Standard des méthodes d&#39;hybridation in situ prendre plus de temps pour sceller les diapositives avec du parafilm, vernis à ongles, et d&#39;autres moyens, où les diapositives prennent beaucoup d&#39;espace; (3) Il est trèssensibles pour les transcriptions de faible abondance en raison de sulfate de dextran et la solution de Denhardt dans le tampon d&#39;hybridation 2,13; (4) L&#39;approche d&#39;imagerie fond noir donne des images à contraste élevé en raison de prendre des photos sous un éclairage sur fond noir sur un microscope à dissection 4,5. En outre, il permet la détection sensible de petits changements dans l&#39;expression des gènes, comme dans l&#39;expression des gènes dépendant de l&#39;activité pour identifier les régions cérébrales activées lors de la perception spécifiques et la production de comportements spécifiques 19. Les limitations relatives à la non-radioactifs protocoles sont que celui-ci sont plus claires dans la résolution cellulaire et l&#39;emplacement de l&#39;ARNm dans la cellule, et en travaillant avec l&#39;émulsion est très sensible à la manipulation et la lumière. Il est possible de combiner notre méthode avec d&#39;autres méthodes, telles que la non-radioactifs hybridation in situ pour étiqueter l&#39;expression des ARNm de plus d&#39;un gène dans le tissu même 10,17,20. Il peut être combiné avec immunocytochimied&#39;étiqueter les deux ARN et expression de la protéine sur le même échantillon, afin de co-localiser l&#39;ARNm avec certains types de cellules 10. Les modifications du protocole nécessaires à ces expériences de double marquage sont décrites dans les références citées. En résumé, notre approche facilite la compréhension du calendrier et de localisation cellulaire de l&#39;expression des gènes dans le but de comprendre l&#39;organisation la région, l&#39;activité des tissus fonctionnels, et la fonction des gènes.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> L&#39;anhydride acétique </td><td> VWR </td><td> MK242002 </td><td></td></tr><tr><td> Chloroforme </td><td> VWR </td><td> BDH1109 </td><td></td></tr><tr><td> Acétate de violet de crésyl </td><td> Sigma </td><td> C5042 </td><td></td></tr><tr><td> Cryostat </td><td> Thermo Scientific </td><td> Microm HM550 </td><td></td></tr><tr><td> Formamide déionisé </td><td> Sigma </td><td> F9037 </td><td></td></tr><tr><td> TNT </td><td> Promega </td><td> P1171 </td><td> 100 mM </td></tr><tr><td> EDTA </td><td> Sigma </td><td> ED </td><td></td></tr><tr><td> Intégration moule </td><td> VWR </td><td> 15160-215 </td><td></td></tr><tr><td> Fisher marque SuperFrost Plus diapositive </td><td> Fisher Scientific </td><td> 22-034-979 </td&gt; <td></td></tr><tr><td> Formaldéhyde </td><td> VWR </td><td> BDH0506-4LP </td><td></td></tr><tr><td> Le formamide </td><td> Sigma </td><td> F7508 </td><td></td></tr><tr><td> Geneclean kit </td><td> Q-Bio gène </td><td> 1001-200 </td><td></td></tr><tr><td> Kodak BioMax MR du film </td><td> Sigma </td><td> Z350370 </td><td></td></tr><tr><td> Kodak NTB Emulsion </td><td> Carestream Health </td><td> 8895666 </td><td></td></tr><tr><td> Kodak Professional développeur D19 </td><td> Kodak </td><td> 1462593 </td><td></td></tr><tr><td> Kodak professionnel Fixer </td><td> Kodak </td><td> 1971746 </td><td></td></tr><tr><td> β-mercaptoéthanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Huile minérale </td><td> VWR </td><td> IC15169491 </td><td></td></tr><tr><td> NaOH </td><td> VWR </td><td> SX0600-1 </td><td></td></tr><tr><td> Paraformaldéhyde </td><td> Sigma </td><td> 76240 </td><td></td></tr><tr><td> Un Poly </td><td> Invitrogen </td><td> POLYA.GF </td><td></td></tr><tr><td> rATP </td><td> Promega </td><td> P1132 </td><td> 10 mM </td></tr><tr><td> rCTP </td><td> Promega </td><td> P1142 </td><td> 10 mM </td></tr><tr><td> rGTP </td><td> Promega </td><td> P1152 </td><td> 10 mM </td></tr><tr><td> RNasin </td><td> Promega </td><td> N2111 </td><td> 40Units/μl </td></tr><tr><td> S 35 UTP </td><td> PerkinElmer </td><td> NEG039C001MC </td><td></td></tr><tr><td> Sécurité-Solve solution </td><td> Sécurité Résolvez recherche Products International </td><td> 111177 </td><td></td></tr><tr><td> Acétate de sodium </td><td> Sigma </td><td> S7899 </td><td> 3M </td></tr><tr><td> Phosphate de sodium dibasique </td><td> Sigma </td><td> S3264 </td><td></td></tr><tr><td> Phosphate monosodique </td><td> Sigma </td><td> S3139 </td><td></td></tr><tr><td> SP6ARN polymérase </td><td> Promega </td><td> P1085 </td><td></td></tr><tr><td> Coloration grille métallique </td><td> Sciences Electron Microscopy </td><td> 70312-54 </td><td></td></tr><tr><td> T7 RNA polymérase </td><td> Promega </td><td> P2075 </td><td></td></tr><tr><td> Tissue-Tek octobre </td><td> Sakura </td><td> 4583 </td><td></td></tr><tr><td> Un tampon de transcription 5x </td><td> Promega </td><td> P1181 </td><td></td></tr><tr><td> Triéthanolamine </td><td> VWR </td><td> IC15216391 </td><td></td></tr><tr><td> Tris-HCl (1 M, pH 8,0) </td><td> VWR </td><td> 101449-446 </td><td></td></tr><tr><td> ARNt </td><td> Roche </td><td> 10109509001 </td><td></td></tr></tbody></table> Solutions: <ol><li> Réactifs pour la fabrication de ribosondes: 1,5 matrice d&#39;ADN ul (0,2-0,3 pg / pl), 2 pl de tampon de transcription 5x optimisé, 1 pl de la TNT 100mm (livré avec la polymérase de Promega, mélangebien à la température ambiante), 0,3 pi de RNasin (40 unités / pl), 1,5 pi de solution AGC mélange ribonucléotide, 3,5 l de S 35 UTP, et 1 polymérase ARN ul. Apportez jusqu&#39;à 10 pi avec une eau sans nucléase. </li><li> Solution AGC mélange ribonucléotide: mélanger des quantités égales de 10 mM ATP, GTP et CTP ensemble. </li><li> Solution d&#39;acétate de sodium pour EtOH la précipitation pour éliminer gratuitement S 35 UTP: 40 pl de RNase et DNase eau libre, 5 ul d&#39;acétate de sodium 3M, et 125 ul d&#39;EtOH à 100%. </li><li> Tampon d&#39;hybridation (10ml stock): 5 ml de formamide désionisée 100%, 600 pi de NaCl 5M, 1M Tris-HCl (pH = 8,0), 240 ul d&#39;EDTA 0,5 M (pH = 8,0), 100 pi de solution de 100x Denhart, 100 pi de 1 million de TNT, 250 pi de 20mg/ml ARNt, 125 pi de 20 mg / ml de poly A, et 1 g de sodium de sulfate de dextrane. Ajouter de l&#39;eau traitée au DEPC à porter le volume total à 10 ml. Agiter vigoureusement et incuber à 55 ° C jusqu&#39;à ce que tout le sulfate de sodium dextran est dissoute. Stockez le tampon d&#39;hybridation dans-20 ° C, ce qui est bon pour environ 6 mois. </li><li> 4% solution tamponnée paraformaldéhyde: ajouter 40 g de paraformaldéhyde en ml d&#39;eau distillée 760 dans un ballon désigné pour paraformaldéhyde, la chaleur à 50 ° C sur une plaque chaude, tout en agitant. Ajouter 320 ul de NaOH 10N pour aider à dissoudre le paraformaldéhyde. Après la dissolution (~ 10 min), ajouter 100 ml de PBS 10X, et porter le volume avec de l&#39;eau distillée jusqu&#39;à 1 litre. Remuer et chauffer la solution jusqu&#39;à ce que le paraformaldéhyde est dissous. Le pH doit être de 7,4. </li><li> L&#39;acétylation de tampon: 13,6 ml de triéthanolamine plus 2,52 ml d&#39;anhydride acétique dans 1 litre d&#39;eau distillée. </li><li> 20x PESS solution: 3M NaCl, 200 mM NaH 2 PO 4-H 2 O, et 200 mM d&#39;EDTA dans l&#39;eau distillée. Ajuster la solution à pH 7,4 avec NaOH 10N. </li><li> 10x tampon PBS: 80 g de NaCl, 2,0 g de KCl, 14,4 g de Na 2 HPO 4, et 2,4 g de KH 2 PO 4 dans l&#39;eau distillée. Ajuster la solution à pH 7,0 et porter le volume total à 1 Liter. </li><li> Deuxième solution de lavage: 0,1% β-mercaptoéthanol et 50% de formamide dans 2x SSPE </li><li> Crésyl solution d&#39;acétate de violet: 3% d&#39;acétate de crésyl violet dans l&#39;eau du robinet (eau distillée empêche la coloration bonne), dissous une nuit sous agitation dans un flacon à la température ambiante. Filtrez avec aspiration par le vide à travers un papier filtre Whatman 1mm et Büchner. </li></ol>

radioactive in situ hybridization for detecting diverse gene expression patterns in tissue

Connaissant le calendrier, le niveau, la localisation cellulaire, et le type de cellules qu&#39;un gène est exprimé dans contribue à notre compréhension de la fonction du gène. Chacune de ces fonctionnalités peut être accompli avec l&#39;hybridation in situ d&#39;ARNm dans les cellules. Nous présentons ici une méthode radioactive dans l&#39;hybridation in situ modifié à partir de Clayton et al. (1988) 1 qui a été travaillé avec succès dans notre laboratoire depuis de nombreuses années, en particulier pour les adultes cerveaux vertébrés 2-5. Les longues complémentaires ARN (ARNc) sondes à la séquence cible permet la détection de transcrits de faible abondance 6,7. Incorporation de nucléotides radioactifs dans les sondes d&#39;ARNc permet en outre la sensibilité de détection de transcrits de faible abondance et des analyses quantitatives, soit sensible à la lumière par un film radiographique ou recouverte d&#39;une émulsion sur le tissu. Ces méthodes de détection de fournir un enregistrement de longue durée de l&#39;expression du gène cible. Par rapport à la sonde non radioactive méthods, comme DIG-étiquetage, la méthode d&#39;hybridation sonde radioactive ne nécessite pas d&#39;amplification des mesures multiples en utilisant HRP-anticorps et / ou TSA kit pour détecter des transcrits de faible abondance. Par conséquent, cette méthode fournit une relation linéaire entre l&#39;intensité du signal et des quantités d&#39;ARNm ciblés pour l&#39;analyse quantitative. Il permet de traiter simultanément les diapositives 100-200. Il fonctionne bien pour les différents stades de développement d&#39;embryons. La plupart des études de développement de l&#39;expression des gènes utiliser des embryons entiers et non-radioactifs approches 8,9, en partie parce que les tissus embryonnaires est plus fragile que le tissu adulte, avec moins de cohésion entre les cellules, ce qui rend difficile de voir les frontières entre les populations de cellules avec des coupes de tissus. En revanche, notre approche radioactifs, en raison de la plus grande plage de sensibilité, est en mesure d&#39;obtenir un contraste plus élevé dans la résolution de l&#39;expression génique entre les régions des tissus, ce qui rend plus facile à voir les limites entre les populations. En utilisant cette méthode, les chercheurs pourraient révéler lasignification possible d&#39;un gène nouvellement identifié, et en outre prédire la fonction du gène d&#39;intérêt.

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

Ce protocole est utilisé avec succès pour détecter quantitativement les niveaux et les schémas spatiaux de l&#39;expression des ARNm de multiples types de tissus à travers les espèces de vertébrés. La méthode peut détecter des transcrits de faible abondance et permet de traiter des centaines de diapositives simultanément. Nous présentons ce protocole en utilisant le profil d&#39;expression de la formation du cerveau embryonnaire aviaire comme un exemple.

Radioactif In situ D&#39;hybridation pour détecter divers profils d&#39;expression génique dans les tissus

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

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Biology

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

15.5K vues.  Duke University. Ce protocole est utilisé avec succès pour détecter quantitativement les niveaux et les schémas spatiaux de l'expression des ARNm de multiples types de tissus à travers les espèces de vertébrés. La méthode peut détecter des transcrits de faible abondance et permet de traiter des centaines de diapositives simultanément. Nous présentons ce protocole en utilisant le profil d'expression de la formation du cerveau embryonnaire aviaire comme un exemple.

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

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.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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

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Biologie

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.

Non radioactifs In situ</emProtocole d&#39;hybridation> Applicable pour l&#39;épinette de Norvège et d&#39;un éventail d&#39;espèces végétales

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

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.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

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.

Un débit élevé In situ</emMéthode d&#39;hybridation> pour caractériser les modes d&#39;expression d&#39;ARNm dans les fœtus de souris tractus urogénital

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.

Analyse de la transcription génique unique cellule par l&#39;ARN fluorescent<em&gt; In Situ</emHybridation&gt; (FISH)

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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<em&gt; Saccharomyces cerevisiae</em

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

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.

Haute Résolution Mont entier<em&gt; In Situ</em&gt; Hybridation dans les embryons de poisson zèbre pour étudier l&#39;expression des gènes et la fonction

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.

MicroRNA<em&gt; In situ</em&gt; Hybridation pour les tissus rénaux fixés au formol

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