Name: quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation
Uploaded: 2014-11-26T14:00:00.000+00:00
Duration: 12 min 42 s
Description: Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. Probe Vorbereitung <ol><li> Erhalten mehrere einzigartige Oligonukleotide in Xist (20-30 Nukleotide Länge Oligonukleotide, 63-65 ° C Schmelztemperatur, Tabelle 1) mit einem 5&#39;-Amino Änderung und Aussetzung im Wasser. </li><li> Pool äquimolare Mengen von Oligonukleotiden mit 5&#39;-Amino-Modifikation (insgesamt 4,5 ug) und Etikett mit aminreaktiven fluoreszierenden Farbstoff nach den Anweisungen des Herstellers. <ol><li> Lösen Sie den gepoolten Oligonukleotide in letzten 5 ul Nuklease-freies Wasser und fügen Sie 3 ul 1 M Natriumbicarbonat mit dem Oligonukleotid-Lösung. </li><li> Add 2 ul DMSO in das Fläschchen mit aminreaktiven Farbstoff und kurz vortexen. </li><li> Mischen Sie die Oligonucleotidlösung mit dem Reaktivfarbstoff in DMSO und Inkubation der Mischung bei Raumtemperatur für 1 Stunde im Dunkeln. </li></ol></li><li> Reinige den fluoreszenzmarkierten Oligonukleotidsonden durch G-25-Säule, gefolgt von Ethanolfällung. <ol><li&gt; Centrifuge G-25-Säule bei 750 g für 1 min, um überschüssige Pufferspeicher zu entfernen. </li><li> In 40 ul Nuklease-freies Wasser auf die in Schritt 1.2.3 hergestellten Markierungsmischung und gelten für die Spalte G-25. Zentrifuge bei 750 × g für 2 min. </li><li> Dann werden 50 &amp; mgr; l Nuklease-freies Wasser, 10 &amp; mgr; l 3 M Natriumacetat (pH 5,2) und 250 &amp; mgr; l Ethanol zu der gereinigten Probe aus Schritt 1.3.2. Bei -80 ° C für 30 Minuten und Zentrifugation bei 21.000 xg für 15 min bei 4 ° C. </li><li> Einmal mit 1 ml 70% Ethanol waschen fällt Oligonukleotide. </li><li> Re-suspendieren in 50 &amp; mgr; l Nuklease-freies Wasser oder TE (10 mM Tris-HCl, pH 7,5; 1 mM EDTA) bei einer Endkonzentration von etwa 10 uM. </li></ol></li><li> Verdünne die fluoreszenzmarkierten Oligonukleotidsonden zu einer Endkonzentration von 250 nM mit Hybridisierungspuffer (10% Formamid; 2 × SSC [300 mM NaCl, 30 mM Natriumcitrat]; 2 mg / ml BSA; 10% Dextransulfat). HINWEIS: Mit dieser schnellen FISH-Protokoll, Oligonukleotid-pRoben Xist wurden entworfen und in einer höheren Konzentration als die normale FISH Protokoll unter Verwendung von Oligonucleotidsonden, um die Inkubationszeit für die Hybridisierung verwendet verkürzen. </li></ol> 2. Schieben Vorbereitung HINWEIS: Die Objektträger für Immun FISH mit embryonalen Stammzellen der Maus (ES) oder Embryoidkörper (EB) vorbereitet werden differenzierenden Zellen 20, 21 entweder durch den Anbau von ihnen direkt auf einem Polylysin behandelten Folie oder mit Cytospin mit Zellsuspension. Hier wird durch Cytospin-Präparat gezeigt. <ol><li> Absaugen Kulturmedium in einer 6-Napf-Schale. Mit PBS (Raumtemperatur), und saugen Sie gründlich waschen. </li><li> 0,5 ml Trypsin in den 6-Well-Platte, Inkubation für 5-10 min bei 37 ° C. </li><li> 5 ml Medium und zerstreuen Zellen durch vorsichtiges Auf- und Abpipettieren mehrmals. </li><li> Übertragungszellen in ein 15 ml-Röhrchen, Zentrifuge für 5 Minuten bei 200 × g bei Raumtemperatur. </li><li> Entsorgen Sie das Medium eind sanft die Zellen in 2 ml PBS. </li><li> Zählen von Zellen unter Verwendung eines Hämocytometers und verdünnte Zellsuspension bei 4 x 10 5 Zellen / ml mit PBS. </li><li> Bauen Sie eine Diashow, einen Filter-Karte und eine Kammer mit einem Clip und legen Sie sie in die entsprechenden Schlitze in dem Rotor der Cytospin. Fügen Sie 250 ul jeder Probe in Schritt 2.6 in jede Kammer der Baugruppen in der Cytospin vorbereitet. Zentrifuge bei 1.500 Upm für 10 min bei Raumtemperatur. </li><li> Füllen 3 Coplin Gläser auf Eis mit 40 ml eiskaltem PBS, CSK-Puffer (10 mM PIPES, pH 6,8, 100 mM NaCl, 300 mM Saccharose; 3 mM MgCl 2) und CSKT Puffer (CSK-Puffer mit 0,5% Triton X-100), jeweils. </li><li> Nachdem der Cytospin beendet ist, die Folie schnell in eiskaltem PBS übertragen und Inkubation für 5 min. </li><li> Die Objektträger in eiskaltes CSK Puffer und Inkubation für 1 min. </li><li> Die Objektträger in eiskaltes CSKT Puffer und Inkubation für 5 min. </li><li> Die Objektträger wieder in eiskaltes CSK buffer und Inkubation für 1 min. </li><li> Die Objektträger in 4% Paraformaldehyd in PBS und Inkubation für 10 min bei Raumtemperatur. </li></ol> 3. Immunfluoreszenz <ol><li> Objektträger in eine Coplin-Gefäß mit PBST (1x PBS mit 0,1% Tween-20) für mehrere Sekunden. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit aus der Folie und legen Sie sie in einem leeren Pipettenspitzen-Box mit Wasser. Überlagern Sie die Folie mit 40 ul Blockierungslösung (1x PBS, 1% BSA, 0,1% Tween-20), sanft legen Sie eine Deckglas, und Inkubation für 10 min bei Raumtemperatur. </li><li> Entfernen Sie das Deckglas, entfernen Sie die Blockierungslösung, und fügen Sie 40 ul der Histone H3K27me3 primären Antikörpers zu einer Konzentration von 1: 500 verdünnt in Blockierungslösung. Legen Sie das Deckglas wieder auf der Folie und Inkubation für 30 min bei Raumtemperatur. HINWEIS: Ein RNase-Inhibitor in der Blockierungslösung könnte hilfreich sein, um RNA in dieser RNA FIS erhaltenH-Protokoll bei polyklonalen Antikörpern eingesetzt werden. </li><li> Wash schieben 2 mal in Coplin Gläser mit PBST gefüllt, je 5 Minuten, leichtes Schütteln beim Waschen. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit und legen Sie die Folie wieder in die Spitze ein. Overlay mit 40 ul des sekundären Antikörpers (1: 500) in Blocking-Lösung verdünnt, setzen Sie ein Deckglas, und Inkubation für 15 min im Dunkeln. Für die folgenden Schritte, halten Sie die Folie in der Dunkelheit zu allen Zeiten. </li><li> In Färbetrog mit PBST, wie in Schritt 3.4 Waschen Sie 2 mal. </li></ol> 4. RNA-FISH mit Oligonukleotidsonden <ol><li> Pre-Aufwärmen eine leere Pipettenspitzen-Box mit Wasser auf dem Boden, um Folien aus dem Trocknen bei 37 ° C für die RNA-FISH in Schritt 4.3 zu verhindern. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit aus der Folie aus Schritt 3.6 und platzieren Sie die Folie in der Pipettenspitzen-Box mit water an der Unterseite. Gib 500 ul FISH Waschpuffer (10% Formamid in 2 × SSC) auf dem Objektträger, Inkubation 5 min bei Raumtemperatur. </li><li> Entfernen FISH Waschlösung, fügen 8 ul von Oligonukleotid-Sonden auf dem Objektträger, legen Sie ein Deckglas, und übertragen Sie die Folie in eine vorgewärmte Pipettenspitzen-Box mit Wasser auf dem Boden; Inkubieren bei 37 ° C für 1-2 Std. </li><li> Platz 3 vorgewärmt Coplin-Glaszylinder in einem Wasserbad bei 37 ° C mit FISH Waschpuffer, FISH Waschpuffer mit DAPI und 2 × SSC. </li><li> Nach 1-2 h Inkubation für RNA FISH, setzen Sie den Schieber in die vorgewärmte FISH Waschpuffer. </li><li> Inkubieren für ein paar Minuten, bis die Deck fällt der Folie. </li><li> Den Objektträger wieder in die Waschpuffer, und weiter Inkubation für 5 min. </li><li> Die Objektträger in einem anderen Coplin-Gefäß mit vorgewärmter FISH Waschpuffer mit 0,5 ng / ml DAPI und Inkubation für 5 min. </li><li> Die Objektträger in einem anderen Coplin Glas mit vorgewärmten 2x SSC und Inkubation für 5 min. </li><li> Legen Sie die Dias schräg so dass Restpuffer zu fließen auf der Rutsche. Wischen Sie vorsichtig die überschüssige Flüssigkeit und legen Sie den Rückfall in einem trockenen Spitze Box. In 4 ul Antifade mit DAPI auf den Objektträger und setzen Sie ein Deckglas. </li><li> Deckglasränder mit Nagellack und beobachten unter dem Mikroskop. Der Schieber kann bei 4 ° C im Dunkeln für einige Monate gelagert werden. </li></ol>

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No conflicts of interest declared.

In diesem Papier haben wir eine schnelle Immun FISH-Protokoll, das weniger als 5 Stunden dauert, Präparat, Xist-RNA FISH und Immunfluoreszenz für H3K27me3 abzuschließen präsentiert. Im Vergleich mit der allgemeinen Immun FISH Ansatz für Xist-RNA-Erkennung, die in der Regel braucht Inkubation über Nacht für die RNA-FISH, dieses Protokoll nicht nur deutlich reduziert Zeitaufwand, sondern verbessert auch die Empfindlichkeit der Immun-FISH mit Oligonukleotid-Sonden. Seit Xist ist hoch wä...

Säugetier X-Chromosom Inaktivierung (XCI) ist eine Strategie, um die Unwucht in X-verknüpften Gens Dosis zwischen XX und XY, wobei eines der beiden X-Chromosomen in Weibchen transkriptionell inaktivierten 1 kompensieren. X-Chromosom-Inaktivierung ist eine große Modellsystem für lange nicht-kodierende RNA (lncRNA) Forschung. X-Chromosom-Inaktivierung von mehreren lncRNAs geregelt und wurde ausgiebig in den letzten Jahrzehnten untersucht, um die Übersprechmechanismen zwischen lncRNAs, Transkription, Chromatinstruktur und Kernorganisation 2, 3 aufzudecken. Die X-Inaktivierung Zentrum (XIC) auf dem X-Chromosom lokalisiert ist eine komplexe genetische Locus einer Reihe von Genen, die Herstellung nicht-kodierenden RNAs 4 besteht. X-inaktiv spezifische Transkript (Xist) lncRNA in eutherian Säugetieren ist eine solche lncRNA, die eine entscheidende Rolle bei der X-Chromosom-Inaktivierung 5,6 spielt. Xist Transkripte umgeben den Ort der futur Xi bis X-Chromosom-Inaktivierung zu initiieren, und erscheinen wie eine Wolke, wenn visualisiert mittels RNA FISH; Diese Formation wird als &quot;Xist Cloud&quot; 7 bezeichnet. Seit Xist-RNA interagiert mit verschiedenen Chromatin-modifizierende Enzyme, wird Co-Lokalisation der Xist Wolken mit verschiedenen epigenetische Modifikationen für stille Chromatin und repressive Transkription während X-Chromosom Inaktivierung 8 beobachtet. Beispielsweise interagiert Xist-RNA mit polycomb repressive komplexen 2 (PRC2), die für H3K27me3 verantwortlich ist und induziert eine repressive Chromatin Zustand 9. Das Auftreten der Xist Wolke am Xi und seine Co-Lokalisierung mit der intensiven H3K27me3 Änderung stellt eine fakultative Heterochromatin Landschaft des Xi 10,11. Zytogenetischen Techniken, wie DNA / RNA-FISH haben einen langen Weg von der traditionellen Methode mit radioaktiv markierten Sonden 12 auf die jüngsten und modernsten Techniken mit verbesserter Empfindlichkeit und kommenFluoreszenz-Bildgebung mit mehreren Oligonukleotidsonden 13,14. DNA / RNA-FISH in Verbindung mit Immunfluoreszenz wurde routinemäßig als zytologische Werkzeug benutzt, um Raum-Zeit-Atom-Organisation, RNA-Lokalisierung, Chromatinstruktur und Modifikationen zu verstehen. Die Standardsonde Vorbereitung RNA FISH beinhaltet die Verwendung von Plasmid oder bakteriellen künstlichen Chromosoms (BAC) Klonen und deren anschließende Markierung entweder mit Nick-Translation oder Random-Priming-15. Fast 70% der Gene in Mäusen und 40% der Gene in Menschen zeigen jedoch eine Überlappung von Sense- und Antisense-Transkripte 16, daher um Sense- und Antisense-Transkripte unterscheidet die eine strangspezifische FISH-Verfahren. In vitro transkribierte RNA-Sonden (Ribosonde ) werden oft für strangspezifischen RNA-FISH 17,18 verwendet; Dies ist jedoch mit Herstellung eines Plasmid-Klon oder PCR-Produkt mit T7, SP6 oder T3-Promotor und Synthetisieren Ribosonden. Außerdem Ribosonden von genom abgeleitetic DNA oder cDNA enthalten oft unspezifische Regionen und repetitiven Elementen, was zu hohen Hintergrundgeräuschen führen. Ein weiteres Problem ist, dass Ribosonden, die einige hundert Nukleotide lang sind, und enthalten mehrere Fluorophore, nicht effizient in den Zellkern einzudringen. Um dies zu umgehen, wurden mehrere kürzere Oligonukleotidsonden mit einem einzigen Fluorophor am Ende markierten entwickelt worden, die eine gute Empfindlichkeit, einheitliche Signalstärke und der Leichtigkeit der Reinigung und Handhabung von 14 haben. Darüber hinaus sind DNA-Oligonukleotide in der Regel stabiler als RNA. Wir verwendeten eine ähnliche Strategie unter Verwendung von Oligonukleotiden in Xist RNA FISH 19 mit Immunfluoreszenz die epigenetische Dynamik des Xi durch Xist lncRNA während des Prozesses der X-Chromosom Inaktivierung induziert verstehen. Dieses Protokoll beschreibt die Erstellung von Oligonukleotid-Sonden und die richtige Vorbereitung der Zellen, sowie die Verwertung von Immunfluoreszenz und RNA-FISH. Xist-RNA-FISH mit mehreren oligonucleotides ist kostensparenden Lösung in den langfristigen, wenn Xist RNA FISH wird routinemäßig in einem Labor durchgeführt wird. Diese Technik kann verwendet werden, um lncRNAs in Zellen zu identifizieren und gleichzeitig die Abbildung ihrer Co-Lokalisierung mit epigenetische Modifikationen oder Faktoren werden. Ein großer Vorteil des Protokolls ist die Fähigkeit, leicht zu modifizieren, seine Forschungsinteressen gerecht zu werden.

<table><tbody><tr><td>oligonucleotides with 5&amp;rsquo;-amino modification</td><td>IDT</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Reactive Dye</td><td>Life Technologies</td><td>A32750</td><td></td></tr><tr><td>MicroSpin G-25 columns</td><td>GE Healthcare</td><td>27-5325-01</td><td></td></tr><tr><td>Cytospin 2</td><td>Shandon</td><td>59900102</td><td></td></tr><tr><td>Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td><td>Roche</td><td>10715859103</td><td></td></tr><tr><td>Histone H3K27me3 antibody</td><td>Active Motif</td><td>61017</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td><td>Life Technologies</td><td>A21424</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36931</td><td></td></tr></tbody></table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

Kombinieren RNA Fluoreszenz in situ Hybridisierung (FISH) mit Immunfluoreszenz (Immuno-FISH) schafft eine Technik, die auf der Ebene einzelner Zellen verwendet werden können, um die räumliche Dynamik RNA Lokalisierung mit gleichzeitige Einblick in die Lokalisierung von Proteinen, epigenetische Modifikationen und andere Details zu detektieren die durch Immunfluoreszenz markiert werden kann. X-Chromosom-Inaktivierung ist ein Paradigma für lange nicht-kodierende RNA (lncRNA) vermittelte Gen-Silencing. X-inaktiven spezifischen Transkripts (Xist) lncRNA Ansammlung (genannt Xist Wolke) an einem der beiden X-Chromosomen in weiblichen Säugern ist ein kritischer Schritt, um X-Chromosom Inaktivierung initiieren. Xist-RNA direkt oder indirekt in Wechselwirkung mit verschiedenen Chromatin-modifizierende Enzyme und stellt verschiedene epigenetischen Landschaften in den inaktiven X-Chromosom (Xi). Eine bekannte epigenetischen Kennzeichen der Xi ist der Histone H3 trimethyl-Lysin-27 (H3K27me3) Änderung. Hier haben wir eine einfache und schnelle Immun FISH beschreibenProtokoll zum Nachweis von Xist-RNA mit RNA-FISH mit mehreren Oligonukleotid-Sonden in Verbindung mit Immunfluoreszenz von H3K27me3, um die Lokalisierung von Xist-RNA und die damit verbundenen epigenetische Veränderungen zu untersuchen. Verwendung von Oligonucleotidsonden zu einer kürzeren Inkubationszeit und empfindlicher Nachweis von Xist RNA im Vergleich zu in vitro transkribierte RNA-Sonden (Ribosonden). Dieses Protokoll stellt ein leistungsfähiges Werkzeug für das Verständnis der Dynamik von lncRNAs und die damit verbundenen epigenetische Modifikation der Chromatinstruktur, Kern Organisation und Transkriptionsregulation.

Repräsentative Bilder der schnellen Immun FISH in 1A gezeigt. Co-Lokalisation der Xist RNA Wolke und H3K27me3 Signal auf dem Xi in differen weiblichen Zellen nachgewiesen. Am Tag 12 nach Differenzierung, mehr als 90% des EB-Zellen hatte eine Xist Wolke (1B). (; 150 97%, n = 1C) kurze Oligonucleotidsonden effizient in den Zellkern durchdrungen wird, was zu einer Visualisierung mit Xist RNA fast alle H3K27me3 Signale kolokalisiert. <p class="jove_content" fo:keep-tog...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Schnell Fluorescent<em&gt; In-situ-</em&gt; Hybridisierung Protokoll für Xist-RNA in Kombination mit Immunfluoreszenz von Histonmodifikationen in X-Chromosom-Inaktivierung

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.9K Aufrufe.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

Serie: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Das Verständnis frühen Organogenese das vereinfachte<em&gt; In-situ-</em&gt; Hybridisierung Protokoll<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Forschung

Entwicklungsbiologie

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Fluoreszenz In Situ Hybridisierung (FISH) verwenden, um den Zustand der Arm Zusammenhalt im Prometaphase und Metaphase überwachen ich Drosophila Eizellen

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biologie

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Erforschung von X-Chromosomen-Aberrationen in Ovarialzellen durch Fluoreszenz-in-situ-Hybridisierung

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

Genetik

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Die Kombination von Doppel-Fluoreszenz<em&gt; In Situ</em&gt; Die Hybridisierung mit Immunmarkierung für Nachweis der Expression von drei Gene in Mäusehirnschnitten

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Neurowissenschaften

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Quantitative Fluoreszenz In Situ Hybridisierung (FISH) und Immunfluoreszenz (IF) spezifischer Genprodukte in KSHV-infizierten Zellen

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Immunologie und Infektion

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Whole Berg RNA Fluorescent<em&gt; In situ</em&gt; Hybridisierung<em&gt; Drosophila</em&gt; Embryonen

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Kombinierte DNA-RNA-Leuchtstoff<em&gt; In situ</em&gt; Hybridisierung (FISH) zu X-Chromosom-Inaktivierung in Differenzierte Weiblich embryonalen Stammzellen der Maus Studieren

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Schnell Fluorescent

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Das Verständnis frühen Organogenese das vereinfachte

Fluoreszenz In Situ Hybridisierung (FISH) verwenden, um den Zustand der Arm Zusammenhalt im Prometaphase und Metaphase überwachen ich Drosophila Eizellen

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

Erforschung von X-Chromosomen-Aberrationen in Ovarialzellen durch Fluoreszenz-in-situ-Hybridisierung

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Die Kombination von Doppel-Fluoreszenz

Quantitative Fluoreszenz In Situ Hybridisierung (FISH) und Immunfluoreszenz (IF) spezifischer Genprodukte in KSHV-infizierten Zellen

Whole Berg RNA Fluorescent

Kombinierte DNA-RNA-Leuchtstoff

Einfache Methode zur Fluoreszenz DNA