Die Autoren möchten allen Jarvis Labor Mitglieder, die das Protokoll über die Jahre verbessert danken.

<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>

Wir haben nichts zu offenbaren.

Radioaktive in situ-Hybridisierung der mRNA-Expression wird häufig für verschiedene Zwecke, einschließlich der für die Untersuchung regionalen Gewebeorganisation, Zelltypen und Gehirn funktionelle Aktivität 2-5,10,12-14 verwendet. Die spätere Verwendung auf Gene, deren mRNA-Expression im Gehirn ist abhängig von erhöhten neurale Aktivität, die oft als leistungsabhängige Gene oder unmittelbar frühen Gene. Mit diesen Anwendungen hat sich unser Verfahren auf mehrere Arten angewendet worden, darunter in Vögel, Säugetiere (zB menschliche), Fische und Amphibien; in mehreren Geweben, einschließlich Gehirn, Haut und Muskel, und mehrere Altersklassen, einschließlich Jungtiere / Neugeborenen, Jugendlichen , Erwachsene, und hier im ganzen Embryo Abschnitten 2,3,5,15-17. Die besonderen Merkmale unseres Protokolls sind: (1) Es erzeugt ein Gleichgewicht zwischen anatomischen Besonderheit und quantitative Spezifität. Um die Genexpression auf dem Röntgenfilm zu quantifizieren, wir digitale Bilder der Bilder (. Bsp.: Abb. 1A und 1B) nehmen, verwenden Sie die Photoshop (Adobe) Histogramm-Funktion, die Pixeldichte in den Regionen von Interesse zu messen und subtrahieren die Hintergrundwerte auf dem Film außerhalb des Gewebes, aber immer noch auf dem Objektträger 2,4. Um die Expression auf zellulärer Ebene zu quantifizieren, nehmen wir Bilder von Silberkörnern über Zellen bei starker Vergrößerung (40-100X; Abb. 4). Wir verwenden die Schwelle und Messfunktionen der Bild J von Wayne Rasband am NIH, die Anzahl der Silberkörner in Bildes zu zählen, subtrahieren den Hintergrund Anzahl in einem ähnlichen Bereich ohne Zellen auf dem Objektträger, durch die Anzahl der Zellen teilen, die a-Werte von Expression pro Zelle. 4,18 (2) relativ hohen Durchsatz sein kann, damit die Verarbeitung von 100-200 Folien gleichzeitig, durch die dichte Abdichtung durch das Deckglas Mineralölbad angelegt zu erhalten. Standard-in-situ Hybridisierung Methoden länger dauern, um die Folien mit Parafilm, Nagellack und andere Mittel, wo Dias nehmen viel Raum zu versiegeln, (3) Es ist höchstempfindlich für geringe Häufigkeit Transkripte durch Dextransulfat und Denhardt-Lösung im Hybridisierungspuffer 2,13, (4) Die Bildgebungsverfahren im Dunkelfeld liefert Bilder mit hohem Kontrast durch Fotografieren unter Dunkelfeldbeleuchtung auf einem Präpariermikroskop 4,5. Darüber hinaus ermöglicht es empfindliche Detektion von kleinen Veränderungen in der Genexpression, wie in Aktivitäts-abhängigen Genexpression auf bestimmte Hirnregionen während der Wahrnehmung und Produktion von spezifischen Verhaltensweisen 19 aktiviert zu identifizieren. Die Einschränkungen in Bezug auf nicht-radioaktiven Protokolle sind, dass diese deutlicher in zellulärer Auflösung und die Position der mRNA in der Zelle sind, und die Arbeit mit Emulsion ist sehr empfindlich gegenüber Manipulation und Licht. Es ist möglich, unsere Methode mit anderen Methoden, wie nicht-radioaktive in situ-Hybridisierung an mRNA-Expression von mehr als einem Gen in dem gleichen Gewebe 10,17,20 kennzeichnen kombinieren. Es kann mit Immunzytochemie kombiniert werden, beide RNA-und Protein-Expression mit der gleichen Probe zu kennzeichnen, um die Co-Lokalisation der mRNA mit bestimmten Zelltypen 10. Die Protokoll-Modifikationen notwendig für eine solche doppelte Kennzeichnung Experimente sind in den zitierten Referenzen beschrieben. Zusammenfassend ermöglicht unser Ansatz Verständnis der Zeitpunkt und die zelluläre Lokalisation der Genexpression, um das Verständnis Region Organisation, Gewebe funktionelle Aktivität und Genfunktionen.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Essigsäureanhydrid </td><td> VWR </td><td> MK242002 </td><td></td></tr><tr><td> Chloroform </td><td> VWR </td><td> BDH1109 </td><td></td></tr><tr><td> Kresylviolett Acetat </td><td> Sigma </td><td> C5042 </td><td></td></tr><tr><td> Kryostat </td><td> Thermo Scientific </td><td> Microm HM550 </td><td></td></tr><tr><td> Deionisiertem Formamid </td><td> Sigma </td><td> F9037 </td><td></td></tr><tr><td> DTT </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> Einbettform </td><td> VWR </td><td> 15160-215 </td><td></td></tr><tr><td> Marke Fisher Superfrost plus Rutsche </td><td> Fisher Scientific </td><td> 22-034-979 </td&gt; <td></td></tr><tr><td> Formaldehyd </td><td> VWR </td><td> BDH0506-4LP </td><td></td></tr><tr><td> Formamid </td><td> Sigma </td><td> F7508 </td><td></td></tr><tr><td> Kit GENECLEAN </td><td> Q-Bio-Gen </td><td> 1001-200 </td><td></td></tr><tr><td> Kodak Biomax MR 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 Entwickler D19 </td><td> Kodak </td><td> 1462593 </td><td></td></tr><tr><td> Kodak Professional Fixer </td><td> Kodak </td><td> 1971746 </td><td></td></tr><tr><td> β-Mercaptoethanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Mineralöl </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> Paraformaldehyd </td><td> Sigma </td><td> 76240 </td><td></td></tr><tr><td> Poly-A </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> Sicherheits-Solve-Lösung </td><td> Sicherheit Solve Research Products International </td><td> 111177 </td><td></td></tr><tr><td> Natriumacetat </td><td> Sigma </td><td> S7899 </td><td> 3M </td></tr><tr><td> Dinatriumhydrogenphosphat </td><td> Sigma </td><td> S3264 </td><td></td></tr><tr><td> Mononatriumphosphat </td><td> Sigma </td><td> S3139 </td><td></td></tr><tr><td> SP6RNA-Polymerase </td><td> Promega </td><td> P1085 </td><td></td></tr><tr><td> Die Färbung Metallgestell </td><td> Electron Microscopy Sciences </td><td> 70312-54 </td><td></td></tr><tr><td> T7-RNA-Polymerase </td><td> Promega </td><td> P2075 </td><td></td></tr><tr><td> Tissue-Tek Oktober </td><td> Sakura </td><td> 4583 </td><td></td></tr><tr><td> 5x Transkription Puffer </td><td> Promega </td><td> P1181 </td><td></td></tr><tr><td> Triethanolamin </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> tRNA </td><td> Roche </td><td> 10109509001 </td><td></td></tr></tbody></table> Lösungen: <ol><li> Reagenzien für die Herstellung von Ribosonden: 1,5 ul DNA-Template (0,2-0,3 ug / ul), 2 ul 5x optimierte Transkription Puffer, 1 ul 100 mM DTT (kommt mit Polymerase von Promega, mixauch bei Raumtemperatur), 0,3 ul RNasin (40 Einheiten / ul), 1,5 ul AGC Ribonukleotid-Mix-Lösung, 3,5 ul der S 35 UTP, und 1 ul RNA-Polymerase. Bringt bis zu 10 ul mit Nuklease-freiem Wasser. </li><li> AGC Ribonukleotid-Mix-Lösung: Mix gleiche Mengen von 10 mM ATP, GTP und CTP zusammen. </li><li> Natriumacetat-Lösung für EtOH Niederschläge auf freie S 35 UTP entfernen: 40 ul RNase und DNase freies Wasser, 5 ul 3 M Natriumacetat und 125 ul 100% EtOH. </li><li> Hybridisierungspuffer (10ml Lager): 5 ml 100% deionisiertes Formamid, 600 ul 5 M NaCl, 1 M Tris-HCl (pH = 8,0), 240 ul 0,5 M EDTA (pH = 8,0), 100 ul 100x Denharts Lösung, 100 ul 1 M DTT, 250 ul 20mg/ml tRNA, 125 ul 20 mg / ml Poly A, und 1 g Natriumdextransulfat. In DEPC-behandeltem Wasser auf ein Gesamtvolumen von 10 ml zu bringen. Kräftig schütteln und dann inkubieren bei 55 ° C, bis alle Natriumdextransulfat aufgelöst wird. Lagern Sie den Hybridisierungspuffer in-20 ° C, was gut für ~ 6 Monate ist. </li><li> 4% gepuffertem Paraformaldehyd-Lösung: Fügen Sie 40 g Paraformaldehyd in 760 ml destilliertem Wasser in einen Kolben für Paraformaldehyd bezeichnet, Hitze bis 50 ° C auf einer Heizplatte unter Rühren zugeben. Fügen Sie 320 ul von 10N NaOH um das Lösen des Paraformaldehyd. Nach dem Auflösen (~ 10 min), 100 ml 10x PBS, und bringe das Volumen mit destilliertem Wasser bis 1 Liter. Umrühren und erhitzen Sie die Lösung bis Paraformaldehyd gelöst ist. Der pH-Wert sollte 7,4 sein. </li><li> Acetylierung Puffer: 13,6 ml Triethanolamin und 2,52 ml Acetanhydrid in 1 Liter destilliertem Wasser. </li><li> 20x SSPE-Lösung: 3M NaCl, 200 mM NaH 2 PO 4-H 2 O und 200 mM EDTA in destilliertem Wasser. Passen Lösung auf pH 7,4 mit 10 N NaOH. </li><li> 10x PBS-Puffer: 80 g NaCl, 2,0 g KCl, 14,4 g Na 2 HPO 4 und 2,4 g KH 2 PO 4 in destilliertem Wasser. Passen Lösung auf pH 7,0 und bringen Gesamtvolumen auf 1 liter. </li><li> Zweiten Waschlösung: 0,1% β-Mercaptoethanol und 50% Formamid in 2x SSPE </li><li> Kresylviolett-Acetat-Lösung: 3% Kresylviolett Acetat in Leitungswasser (destilliertes Wasser verhindert, dass eine gute Färbung), über Nacht unter Rühren in einem Kolben bei Raumtemperatur gelöst. Filter mit Vakuumabsaugung durch ein 1mm Whatman-Filterpapier und Büchner-Trichter. </li></ol>

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

Die Kenntnis der Zeitpunkt, Stufe, zelluläre Lokalisation und Zelltyp, dass ein Gen, das exprimiert wird, trägt zum Verständnis der Funktion des Gens. Jedes dieser Merkmale kann mit in situ-Hybridisierung an mRNAs in den Zellen erreicht werden. Hier präsentieren wir eine radioaktive in situ Hybridisierung Methode von Clayton et al. (1988) 1, die gearbeitet hat erfolgreich in unserem Labor seit vielen Jahren, vor allem für erwachsene Wirbeltieren Gehirne 5.2 geändert. Die langen komplementärer RNA (cRNA) Sonden an die Zielsequenz erlaubt die Detektion von niedriger Häufigkeit Transkripte 6,7. Der Einbau von radioaktiven Nukleotiden in die cRNA-Sonden können zur weiteren Nachweisempfindlichkeit geringer Menge Transkripte und quantitativen Analysen, entweder durch lichtempfindlichen Röntgenfilm oder Emulsion beschichteten über das Gewebe. Diese Nachweisverfahren eine langfristige Aufzeichnung der Expression des Zielgens. Im Vergleich zu nicht-radioaktive Sonde Meth-Methoden, wie z. B. DIG-Etikettierung ist die radioaktive Sonde Hybridisierungsverfahren nicht mehrere Schritte Amplifikation unter Verwendung von HRP-Antikörper und / oder TSA-Kit geringer Menge Transkripte zu erfassen. Daher stellt dieses Verfahren eine lineare Beziehung zwischen der Signalintensität und gezielte mRNA-Mengen für die quantitative Analyse. Es erlaubt die Verarbeitung von 100 bis 200 Objektträger gleichzeitig. Es funktioniert gut für die verschiedenen Entwicklungsstadien der Embryonen. Die meisten Entwicklungsstudien der Genexpression nutzen ganzen Embryonen und nicht-radioaktive Ansätze 8,9, zum Teil weil embryonalem Gewebe ist zerbrechlicher als erwachsenem Gewebe, mit weniger Zusammenhalt zwischen den Zellen, was es schwierig macht Grenzen zwischen Zell-Populationen mit Gewebeschnitten zu sehen. Im Gegensatz dazu ist unsere radioaktive Ansatz, aufgrund der größeren Auswahl an Sensibilität, in der Lage, einen höheren Kontrast in der Auflösung der Genexpression zwischen Gewebe Regionen zu erhalten, wodurch es leichter, Grenzen zwischen den Populationen zu sehen. Mit dieser Methode konnten die Forscher zeigen, diemögliche Bedeutung eines neu identifizierte Gen, und ferner vorherzusagen die Funktion des Gens von Interesse.

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

Dieses Protokoll wird erfolgreich eingesetzt, um quantitativ zu detektieren Ebenen und räumliche Muster der mRNA-Expression in mehreren Gewebetypen über Wirbeltierarten. Das Verfahren lässt sich erkennen geringer Menge Transkripte und ermöglicht die Verarbeitung von Hunderten von Dias gleichzeitig. Wir präsentieren dieses Protokoll mit Expression Profiling der aviären embryonalen Gehirn Bildung als Beispiel.

Radioaktiv In-situ- Hybridisierung zum Nachweis Diverse Genexpressionsmuster 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

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JoVE Journal

Biology

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

15.5K Aufrufe.  Duke University. Dieses Protokoll wird erfolgreich eingesetzt, um quantitativ zu detektieren Ebenen und räumliche Muster der mRNA-Expression in mehreren Gewebetypen über Wirbeltierarten. Das Verfahren lässt sich erkennen geringer Menge Transkripte und ermöglicht die Verarbeitung von Hunderten von Dias gleichzeitig. Wir präsentieren dieses Protokoll mit Expression Profiling der aviären embryonalen Gehirn Bildung als Beispiel.

Serie: 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.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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

Forschung

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.

Nicht radioaktiv In situ Hybridisierung Protocol Einsetzbar für Fichte und einer Reihe von Pflanzenarten

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.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

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.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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.

In vitro Rekonstitution der 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 ...

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.

Ein hoher Durchsatz In situ Hybridisierung Methode zur mRNA Expression Patterns in der Fetal-Maus unteren Urogenitaltrakts Charakterisieren

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 von Einzel-Zell-Gene Transkription durch RNA-Fluorescent<em&gt; In Situ</em&gt; Hybridisierung (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.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<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.

Hohe Auflösung Ganze Berge<em&gt; In Situ</em&gt; Hybridisierung innerhalb Zebrafischembryonen Gene Expression und Funktion zu untersuchen

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; Hybridisierung für Formalin fixiert Nierengewebe

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