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. Préparation de la sonde <ol><li> Obtenir de multiples oligonucléotides uniques dans Xist (oligonucléotides 20-30 nucléotides de longueur, de 63 à 65 ºC température de fusion, tableau 1) avec une modification 5&#39;-amino et de suspendre dans l&#39;eau. </li><li> Piscine quantités équimolaires d&#39;oligonucléotides avec 5&#39;-amino modification (total 4,5 ug) et étiqueter avec colorant fluorescent amine réactive en suivant les instructions du fabricant. <ol><li> Dissoudre les oligonucléotides regroupés dans Final 5 pi d&#39;eau sans nucléase et ajouter 3 pl de 1 M de bicarbonate de sodium à la solution d&#39;oligonucléotide. </li><li> Ajouter 2 ul DMSO pour le flacon de colorant et vortex amine réactive brièvement. </li><li> Mélanger la solution d&#39;oligonucléotides avec le colorant réactif dans du DMSO et on incube le mélange à température ambiante pendant 1 heure dans l&#39;obscurité. </li></ol></li><li> On purifie les sondes oligonucléotidiques marquées par fluorescence par colonne G-25 suivie d&#39;une précipitation à l&#39;éthanol. <ol><li&gt; Centrifugeuse colonne G-25 à 750 x g pendant 1 minute pour éliminer l&#39;excès de tampon de stockage. </li><li> Ajouter de l&#39;eau sans nucléase 40 pi au mélange d&#39;étiquetage produit dans l&#39;étape 1.2.3 et appliquer à la colonne G-25. Centrifuger à 750 g pendant 2 min. </li><li> Ajouter 50 ul d&#39;eau sans nuclease, 10 ul de 3 M d&#39;acétate de sodium (pH 5,2) et 250 ul d&#39;éthanol à la sonde purifiée de l&#39;étape 1.3.2. Conserver à -80 ° C pendant 30 min et centrifuger à 21 000 g pendant 15 min à 4 ° C. </li><li> Laver les oligonucleotides précipitées une fois avec 1 ml d&#39;éthanol à 70%. </li><li> Remettre en suspension dans 50 pi d&#39;eau sans nuclease ou TE (10 mM Tris-HCl, pH 7,5; 1 mM EDTA) à une concentration finale d&#39;environ 10 uM. </li></ol></li><li> Diluer les sondes oligonucléotidiques marquées par fluorescence à une concentration finale de 250 nM avec un tampon d&#39;hybridation (10% formamide; 2x SSC [NaCl 300 mM; citrate de sodium 30 mM]; 2 mg / ml de BSA; 10% de sulfate de dextrane). NOTE: Avec ce protocole FISH rapide, oligonucléotide ppeignoirs dans Xist ont été conçus et utilisés à une concentration supérieure à la normale protocole FISH en utilisant des sondes oligonucléotidiques dans le but de raccourcir le temps d&#39;incubation pour l&#39;hybridation. </li></ol> 2. Préparation de la lame REMARQUE: Les diapositives peuvent être préparé pour immuno-FISH utilisant souches de souris embryonnaires (ES) ou le corps embryoïdes (EB) la différenciation des cellules 20, 21 soit par leur culture directement sur ​​lame de poly-lysine-traité ou en utilisant cytospin avec suspension cellulaire. Ici, la préparation de lame par cytospin se affiche. <ol><li> Aspirer le milieu de culture dans une boîte de 6 puits. Laver avec du PBS (température ambiante), et aspirer à fond. </li><li> Ajouter 0,5 ml de trypsine dans le plat à 6 puits, incuber pendant 5-10 min à 37 ° C. </li><li> Ajouter 5 ml de milieu et de disperser les cellules par pipetage doucement monter et descendre plusieurs fois. </li><li> Transférer les cellules dans un tube de 15 ml, centrifuger pendant 5 min à 200 x g à température ambiante. </li><li> Jeter un moyenD délicatement les cellules en suspension dans 2 ml de PBS. </li><li> Compter les cellules en utilisant un hémocytomètre, et on dilue la suspension de cellules à 4 x 10 5 cellules / ml en PBS. </li><li> Assembler un toboggan, une carte de filtre et une chambre avec un clip et placez-les dans les fentes appropriées dans le rotor de la cytospin. Ajouter 250 pi de chaque échantillon préparé à l&#39;étape 2.6 dans chaque chambre des unités assemblées dans le cytospin. Centrifuger à 1500 rpm pendant 10 min à température ambiante. </li><li> Remplir trois bocaux de Coplin sur la glace avec 40 ml chacun de tampon PBS, CSK glacée (PIPES 10 mM, pH 6,8; NaCl 100 mM; mM de saccharose 300; 3 mM MgCl 2), et la zone tampon CSKT (tampon CSK avec 0,5% de Triton X-100), respectivement. </li><li> Après la cytospin est terminée, transférer rapidement le glissement vers PBS glacé et incuber pendant 5 min. </li><li> Placer les lames dans le tampon CSK glacée et incuber pendant 1 min. </li><li> Placer les lames dans le tampon CSKT glacée et incuber pendant 5 min. </li><li> Transférer le retomber dans glacée CSK buffert et incuber pendant 1 min. </li><li> Transférer la diapositive dans 4% de paraformaldehyde dans du PBS et incuber pendant 10 min à température ambiante. </li></ol> 3. immunofluorescence <ol><li> Placer les lames dans une jarre de Coplin avec du PBST (1x PBS avec 0,1% de Tween-20) pour plusieurs sec. </li><li> Placer les lames à une inclinaison permettant tampon restant à se écouler sur le toboggan. Essuyez soigneusement l&#39;excès de liquide hors de la diapositive et placez-le dans une boîte pipette pointe vide avec de l&#39;eau. Superposez la lame avec 40 ul de la solution de blocage (PBS 1x, 1% de BSA, 0,1% de Tween-20), placer délicatement une lamelle, et incuber pendant 10 min à température ambiante. </li><li> Retirer la lamelle, retirez la solution de blocage, et ajouter 40 ul de l&#39;anticorps primaire Histone H3K27me3 dilué à une concentration de 1: 500 dans une solution de blocage. Placer la lamelle couvre-objet sur la lame arrière et incuber pendant 30 min à température ambiante. REMARQUE: Un inhibiteur de RNase dans la solution de blocage peut être utile pour préserver l&#39;ARN dans cet ARN FISH protocole lorsque les anticorps polyclonaux sont utilisés. </li><li> Laver glisser deux fois dans des bocaux de Coplin remplis avec du PBST, pendant 5 minutes chacune, secouant doucement pendant le lavage. </li><li> Placer les lames à une inclinaison permettant tampon restant à se écouler sur le toboggan. Essuyez soigneusement l&#39;excès de liquide et placer la lame dans la boîte de pointe. Superposition avec 40 pi de l&#39;anticorps secondaire (1: 500) dilué dans une solution de blocage, placez une lamelle, et incuber pendant 15 minutes dans l&#39;obscurité. Pour les étapes suivantes, garder la lame dans l&#39;obscurité à tout moment. </li><li> Laver deux fois en COPLIN bocaux avec du PBST comme dans l&#39;étape 3.4. </li></ol> 4. ARN FISH avec sondes oligonucléotidiques <ol><li> Préchauffer une boîte pipette pointe vide avec de l&#39;eau au fond pour empêcher les glissements de sécher à 37 ° C pour l&#39;ARN FISH à l&#39;étape 4.3. </li><li> Placer les lames à une inclinaison permettant tampon restant à se écouler sur le toboggan. Essuyez soigneusement l&#39;excès de liquide de la lame de l&#39;étape 3.6 et placer la lame dans la boîte pipette pointe avec water au fond. Ajouter 500 ul de tampon de lavage FISH (10% de formamide dans du SSC 2x) sur la lame et incuber pendant 5 minutes à température ambiante. </li><li> Retirer la solution de lavage de FISH, ajouter 8 pi de sondes d&#39;oligonucléotides sur la diapositive, placez une lamelle, et transférer la diapositive à une boîte préchauffé pipette pointe avec de l&#39;eau au fond; incuber à 37 ° C pendant 1-2 h. </li><li> Placez trois bocaux de Coplin pré-chauffé dans un bain d&#39;eau à 37 ° C avec un tampon de lavage de poisson, tampon de lavage avec du DAPI, et 2x SSC. </li><li> Après 1-2 heures d&#39;incubation pour l&#39;ARN FISH, mettre la lame dans le tampon préchauffé lavage FISH. </li><li> Incuber la lame pendant quelques minutes jusqu&#39;à ce que la lamelle se détache de la diapositive. </li><li> Placer la lame de nouveau dans le tampon de lavage, et continuer l&#39;incubation pendant 5 min. </li><li> Transférer la diapositive à l&#39;autre bocal de Coplin avec un tampon de lavage FISH préchauffé avec 0,5 ng / ml DAPI et incuber pendant 5 min. </li><li> Transférer la diapositive à l&#39;autre bocal de Coplin avec 2x SSC préchauffé et incuber pendant 5 kmn. </li><li> Placer les lames à une inclinaison permettant tampon restant à se écouler sur le toboggan. Essuyez soigneusement l&#39;excès de liquide et placer la lame de nouveau dans une zone de la pointe sèche. Ajouter 4 pi de antifade avec DAPI sur la lame et placez une lamelle. </li><li> Sceller la lamelle avec du vernis à ongles et d&#39;observer sous un microscope. Le coulisseau peut être stocké à 4 ° C dans l&#39;obscurité pendant quelques mois. </li></ol>

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No conflicts of interest declared.

Dans cet article, nous avons présenté un rapide protocole immuno-FISH qui prend moins de 5 h pour compléter la préparation des lames, ARN Xist FISH, et l&#39;immunofluorescence pour H3K27me3. En comparaison avec l&#39;approche générale immuno-FISH pour les ARN Xist détection, qui a généralement besoin d&#39;une nuit d&#39;incubation pour l&#39;ARN FISH, ce protocole non seulement réduit considérablement le temps passé, mais améliore également la sensibilité du système immunitaire FISH utilisant des sonde...

Mammalian inactivation du chromosome X (XCI) est une stratégie pour compenser le déséquilibre de dosage de gène lié à l&#39;X entre XX et XY, dans lequel l&#39;un des deux chromosomes X chez les femelles est transcriptionnellement 1 inactivé. Inactivation du chromosome X est un système modèle très longtemps ARN non codant (lncRNA) recherche. Inactivation du chromosome X est réglementée par de multiples lncRNAs, et a été largement étudiée au cours des dernières décennies à découvrir les mécanismes de diaphonie entre lncRNAs, la transcription, la structure de la chromatine et l&#39;organisation nucléaire 2, 3. Le centre d&#39;inactivation X (XIC) situé sur le chromosome X est un locus génétique complexe constitué d&#39;un certain nombre de gènes qui produisent des ARN non codants 4. X-inactive transcrit spécifique (Xist) lncRNA chez les mammifères euthériens est un de ces lncRNA qui joue un rôle crucial dans inactivation du chromosome X 5,6. transcriptions de Xist entourent l&#39;emplacement du future Xi pour lancer inactivation du chromosome X, et apparaît comme un nuage quand visualisées en utilisant l&#39;ARN FISH; cette formation est appelé le &quot;Xist Couverture&quot; 7. Étant donné que l&#39;ARN Xist interagit avec diverses enzymes de modification de la chromatine, la co-localisation des nuages ​​Xist avec différentes modifications épigénétiques de la chromatine et la transcription silencieux répressif est observée au cours de l&#39;inactivation du chromosome X-8. Par exemple, l&#39;ARN Xist interagit avec polycomb complexe répressive 2 (PRC2) qui est responsable de H3K27me3 et induit un état ​​de la chromatine répressive 9. L&#39;apparition du nuage de Xist sur ​​le Xi et son co-localisation avec la modification H3K27me3 intensive représente un paysage de hétérochromatine facultative du Xi 10,11. Techniques cytogénétiques, comme l&#39;ADN / ARN FISH ont parcouru un long chemin de la méthode traditionnelle en utilisant des sondes radiomarquées 12 aux techniques récentes et avancées avec une sensibilité accrue etl&#39;imagerie de fluorescence en utilisant plusieurs sondes oligonucléotidiques 13,14. ADN / ARN FISH couplé avec immunofluorescence a été régulièrement utilisé comme un outil cytologique de comprendre l&#39;organisation spatio-temporelle nucléaire, localisation d&#39;ARN, la structure de la chromatine et modifications. La préparation de sonde la plus standard pour ARN FISH implique l&#39;utilisation de plasmide ou de chromosomes artificiels (BAC) clones bactériens et leur étiquetage ultérieure soit avec translation de coupure ou amorçage aléatoire 15. Cependant, près de 70% des gènes chez la souris et 40% des gènes chez l&#39;homme montrent un chevauchement des sens et antisens transcriptions 16, donc nécessitant une méthode de FISH spécifique de brin afin de distinguer sens et antisens transcriptions. In vitro des sondes d&#39;ARN transcrits (ribosonde ) sont souvent utilisés pour spécifique de brin ARN FISH 17,18; Toutefois, il se agit de la préparation d&#39;un clone de plasmide ou d&#39;un produit PCR avec T7, SP6, ou le promoteur de T3 et ribosondes de synthèse. En outre, des ribosondes dérivé de génomeic ADN ou ADNc contiennent souvent des régions non-spécifiques et les éléments répétitifs, qui se traduisent par un bruit de fond élevé. Un autre problème est que ribosondes, qui sont à quelques centaines de nucléotides de long et contiennent plusieurs fluorophores, ne peuvent pas pénétrer efficacement dans le noyau. Pour éviter cela, plusieurs sondes oligonucléotidiques courtes marqués avec un fluorophore unique à la fin ont été développées qui ont une bonne sensibilité, la puissance du signal uniforme, et la facilité de purification et de manipulation 14. En outre, les oligonucleotides d&#39;ADN sont généralement plus stables que les ARN. Nous avons appliqué une stratégie similaire en utilisant des oligonucléotides ARN Xist dans FISH 19 avec immunofluorescence de comprendre la dynamique de l&#39;Xi épigénétiques induites par Xist lncRNA pendant le processus d&#39;inactivation du chromosome X. Ce protocole décrit la création de sondes oligonucléotidiques et une bonne préparation des cellules, ainsi que l&#39;utilisation de l&#39;immunofluorescence et l&#39;ARN FISH. ARN Xist FISH utilisant oligonucle multiplesotides est approche rentable à long terme si ARN Xist FISH est effectuée régulièrement dans son laboratoire. Cette technique peut être utilisée pour identifier lncRNAs dans les cellules tout en traçant simultanément son co-localisation avec des modifications ou des facteurs épigénétiques. Un avantage majeur du protocole est la possibilité de modifier facilement pour l&#39;adapter à ses intérêts de recherche.

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			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
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			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

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

Combinant ARN hybridation fluorescente in situ (FISH) avec immunofluorescence (immuno-FISH) crée une technique qui peut être utilisé au niveau de la cellule unique pour détecter les dynamiques spatiales de localisation d&#39;ARN avec perspicacité simultanée dans la localisation de protéines, les modifications épigénétiques et autres détails qui peut être mis en évidence par immunofluorescence. Inactivation du chromosome X est un paradigme de longue ARN non codant (lncRNA) silençage génique médiée. transcrit spécifique (Xist) accumulation de lncRNA X-inactive (appelé un nuage Xist) sur l&#39;un des deux chromosomes X chez les femelles-mammifères est une étape critique pour initier inactivation du chromosome X. ARN Xist interagit directement ou indirectement avec diverses enzymes de modification de la chromatine et introduit paysages épigénétiques distinctes au chromosome X inactif (Xi). Une des caractéristiques de l&#39;épigénétique connu Xi est l&#39;histone H3 lysine triméthyl-27 (H3K27me3) modification. Ici, nous décrivons un immuno-FISH simple et rapideprotocole pour la détection de l&#39;ARN Xist en utilisant l&#39;ARN FISH avec plusieurs sondes d&#39;oligonucléotides couplés avec immunofluorescence de H3K27me3 d&#39;examiner la localisation des ARN Xist et des modifications épigénétiques associées. Utilisant des sondes oligonucléotidiques se traduit par un temps d&#39;incubation plus courte et une détection plus sensible de l&#39;ARN Xist rapport au transcrit in vitro des sondes d&#39;ARN (des ribosondes). Ce protocole fournit un outil puissant pour comprendre la dynamique de lncRNAs et sa modification épigénétique associé, la structure de la chromatine, organisation nucléaire et la régulation transcriptionnelle.

Des images représentatives de rapide immuno-FISH sont présentés dans la figure 1A. Co-localisation du nuage et H3K27me3 signaux ARN Xist sur le Xi a été détecté dans la différenciation des cellules femelles. Au jour 12 lors de la différenciation, plus de 90% des cellules EB avait un nuage Xist (figure 1B). Des sondes oligonucléotidiques courtes efficacement pénétré dans le noyau, ce qui conduit à une visualisation de la quasi-totalité des signaux H3K27me3 co-localisée av...

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.

Fluorescent rapide<em&gt; In Situ</em&gt; Protocole hybridation pour ARN Xist combinée avec d&#39;immunofluorescence Histone Modification dans inactivation du chromosome X

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

13.8K vues.  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. 

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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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 ...

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.

Zebrafish entier Mont haute résolution double fluorescent In Situ Hybridation

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescence In situ</emL&#39;hybridation> (FISH) du Protocole dans le sperme humain

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hybridation in situ pour la localisation précise des transcriptions dans les plantes

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

La détection de l&#39;ARN viral par fluorescence<em&gt; In situ</emHybridation&gt; (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Timing chromosome Réplication Combiné avec fluorescent<em&gt; In situ</em&gt; Hybridation

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

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.

Toute Mont ARN fluorescent<em&gt; In situ</emHybridation&gt; d&#39;<em&gt; Drosophila</em&gt; Embryons

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

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHybridation&gt; en embryons entiers de montage et histologie Adapté pour les élasmobranches marins

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...