Les auteurs tiennent à remercier Michael Alifrangis et Ulla Abildtrup pour le génotypage des parasites et Christina Holm pour l&#39;assistance technique excellente. Ce travail a été financé par Howard Hughes Medical Institute (subvention 55.005.511), la Fondation Lundbeck (subvention R9-A840) et par la Fondation Niels Bohr.

1. Génération de fraîchement sélectionnées érythrocytes infectés Pour ce test, les meilleurs résultats sont obtenus en utilisant des cultures fraîchement sélectionnés pour l&#39;expression en surface de la protéine PfEMP1. Dans cette expérience particulière du P. 3D7 lignée falciparum a été sélectionné en utilisant des anticorps spécifiques, comme décrit précédemment 1. Jour 1 <ol><li> Récolte 200 ul de cellules sanguines emballés à partir d&#39;un P. falciparum culture contenant 2-5% IE tardivement par centrifugation à 800 xg pendant 8 min à température ambiante. </li><li> Remettre en suspension le culot dans 2 ml de sang de 37 ° C au chaud solution à 0,75% de gélatine dans un tube de 14 ml stérile et laisser reposer pendant 15-20 min à 37 ° C pour permettre l&#39;IE non infectés et la bague au stade de sédiments. </li><li> Récolter les IE au stade avancé, c&#39;est à dire la phase supérieure, dans un nouveau tube 14 ml et laver deux fois avec 10 ml de 37 ° C chaudes RBC-lavage médias. </li><li> Diluer 50 ul d&#39;anticorps spécifiques anti-PfEMP1 anticorps dans 2 ml de 37 ° C chaudes RBC-lavage médias et filtre stérile. </li><li> Mélanger la chaux stade avancé IE avec l&#39;antisérum dilué stérilisé dans un tube de 14 ml stérile et incuber pendant 30 min à 37 ° C sous agitation douce. </li><li> Centrifuger à 800 xg pendant 8 min à température ambiante, et laver deux fois avec 10 ml de 37 ° C chaudes RBC-lavage médias. </li><li> Lavez 50 pi de la protéine Dynabead Une suspension à deux reprises avec RBC-lavage médias utilisant une DynaMaq-15 aimant. </li><li> Remettre en suspension les Dynabeads dans 300 ul RBC-WASH pour les médias et les ajouter à la chaux stade avancé IE. Incuber pendant 30 min à 37 ° C sous agitation douce. </li><li> Transférer le tube de l&#39;aimant DynaMaq-15. Laissez-le pendant 1-2 min et éliminer tout le liquide. </li><li> Remettre en suspension les Dynabeads dans 4-5 ml de RBC de lavage médias. Transfert à l&#39;arrière du tube sur l&#39;aimant et le laisser pendant 1-2 min avant de retirer tout le liquide. Répéter l&#39;étape de lavage une fois. </li><li> Transférer les billes lavées à un 25 cm 2 f de culture stérilelask contenant 200 ul fraîchement concentré de globules rouges. Ajouter 5-6 ml de Albumax médias dans la culture. Stockez nuit à 5% de CO 2 à 37 ° C. </li></ol> Jour 2 <ol start="12"><li> Supprimer les Dynabeads de la culture lorsque la ré-envahi IE a sélectionné les cellules rouges du sang frais, en transférant toute la culture dans un tube de 14 ml stérile. Placer le tube sur le DynaMaq-15 aimant et laisser reposer pendant 1-2 min. Ensuite, versez tout le retour du liquide dans un flacon de culture. </li><li> La culture pour que les cycles possible jusqu&#39;à une parasitémie de 5-10% et les parasites sont à l&#39;ringstage. </li><li> IE doivent être analysés par FACS pour s&#39;assurer que le phénotype de surface correcte, à savoir l&#39;expression des protéines soit and/orPF11_0008 PFD1235w a été obtenue 1. </li></ol> 2. Préparation des sondes Les sondes antisens d&#39;ARN ont été générés à partir des régions les plus variables du PFD1235w et le var PF11_0008 &lt;/ Em&gt; des gènes de P. l&#39;ADN génomique falciparum 3D7. L&#39;ADN a été amplifié par PCR et cloné dans le vecteur pSPT18 ou 19 pour la transcription, respectivement selon la description du fabricant (Roche). Les sondes (580 paires de bases (pb) et 590 pb de longueur) ont été marqués par la digoxigénine (DIG) ou biotine en utilisant un kit de marquage DIG ARN ou une biotine ARN étiquetage Mix, respectivement. Nous avons confirmé la spécificité des sondes à la fois par des analyses par Northern blot et par une seule partie analyse FISH 1. 3. Frottis mince et fixation des parasites Avant hybridation in situ Jour 1 Toutes les étapes sont réalisées dans un environnement exempt de RNase et tous les réactifs sont sans RNase ou pré-traitées avec diéthyl pyrocarbonate (DEPC) ou RNase Zap (Invitrogen). Les diapositives et les lamelles couvre-objets doivent être nettoyés avec de l&#39;alcool pour enlever la graisse de résidu de fabrication. Il est important d&#39;effectuer le protocole sans aucune pause entre les étapesafin de minimiser le risque de contamination nucléase. <ol><li> Faire un frottis sanguin par la méthode standard propagation 3. Utilisation de la lentille de l&#39;objectif 100x du microscope, compter l&#39;IE et de calculer le pourcentage d&#39;IE dans la culture. Vérifiez que les stades parasitaires sont en synchronie approximative, dans l&#39;anneau et les premiers stades trophozoïtes du cycle de IE. Il s&#39;agit de la pré-réplication, uni-nucléés étapes, exprimant l&#39;ARNm du gène var et adapté à des dosages de cellules simples de transcription POISSONS de ce type. </li><li> Transférer 50 ul de la culture du parasite, à une parasitémie de 5-10%, 1,5 ml d&#39;une RNase-free tube Eppendorf. Centrifuger pendant 1 min à 2.000 tours par minute à température ambiante. </li><li> Retirez le support et ajouter 50 ul de PBS pH 7,2 en eau traitée au DEPC. Agiter doucement le tube. Répétez l&#39;étape sédimentation centrifuge à deux reprises pour laver les cellules libres de médias et les débris cellulaires. </li><li> Faites quatre frottis minces de bonne qualité. Pour chaque frottis, faire un frottis surun 11 mm de diamètre et d&#39;une diapositive 4 Les diapositives de verre soit quatre au total, dans une hotte sans RNase (une étape critique). Vague glisse brièvement en l&#39;air pour sécher. Faire trois lames supplémentaires pour les contrôles négatifs tiroir-une sonde de marquage unique pour tout autre gène pertinent à l&#39;aide d&#39;une sonde spécifique, une autre lame pour le traitement RNase et un troisième chariot contenant IE n&#39;exprimant pas le gène d&#39;intérêt. Laisser les lames sécher sur le banc pendant 10-20 min. </li><li> Fixer les couches minces en ajoutant 60 pl de solution de fixation contenant du paraformaldéhyde à 4% et 5% d&#39;acide acétique glacial dans le puits. Laisser reposer 10 minutes sur le banc à la température ambiante. </li><li> Laver les lames dans un tampon 2x SSPE pendant 5 minutes par agitation douce à température ambiante. Retirez brièvement l&#39;excès de liquide en touchant les puits contenant les cellules délicatement avec un mouchoir en papier. </li><li> Couvrir les lames avec de la pepsine 0,01% à 0,01 M HCl pendant 2 min à 37 ° C (une étape extrêmement critique). Laver les lames en 2x SSPE pendant 5 min à température ambiante. </li><li> Diluer la solution RNase à une concentration finale de 10 pg / ml. Recouvrir le frottis de contrôle négatif avec 60 ul de la solution de RNase. Incuber pendant 30 min à 37 ° C, puis laver les lames en 2x SSPE pendant 5 min à température ambiante. </li><li> Procéder immédiatement à l&#39;étape d&#39;hybridation. </li></ol> 4. L&#39;hybridation des sondes d&#39;ARN aux diapositives fixes Jour 1 <ol><li> Préparer la chambre d&#39;hybridation, comme un ThermoStar 100 HC4 ou une boîte vide pipette pointe mis dans un four propre, par pulvérisation avec de la RNase Zap et essuyant le nettoyer avec un mouchoir en papier. Remplir les canaux de la chambre d&#39;hybridation ou le fond de la boîte à eau traitée au DEPC à faire en sorte que les lames ne sèche pas pendant l&#39;hybridation. Fermer la chambre et préchauffer à 48 ° C. </li><li> Diluer les sondes dans la solution d&#39;hybridation à une concentration finale de 12 ng / pl. Dénaturer la solution de sonde à 65 ° C pendant 5 min dans un bloc de chauffage. Immédiatement refroidir sur glace. </li><li> En bref l&#39;air sécher les lames après le lavage 2x SSPE. Retirez délicatement l&#39;excès de liquide dans le puits avec un mouchoir en papier. </li><li> Ouvrez la chambre d&#39;hybridation et placer les lames dans la chambre. Appliquer une ou deux sondes dans un volume total de 60 ul par puits. Couvrez soigneusement la goutte de liquide avec une lamelle RNase-free à l&#39;aide des pinces stériles. </li><li> Fermer la chambre et l&#39;hybridation des lames à 48 ° C pendant au moins 16 heures pendant la nuit. </li></ol> 5. Conjugaison d&#39;anticorps et d&#39;amplification de fluorescence Jour 2 Les étapes suivantes sont basées sur le CST Plus System Palette de fluorescence de Perkin Elmer. Toutes les étapes sont réalisées à température ambiante. <ol><li> Laver les lames 3 fois dans du tampon TNT pendant 5 min sous agitation douce. </li><li> Bloquer le puits dans 150 ul de tampon TNB pendant 30 min. </li><li> Diluer le conjugué HRP-α-DIG anticorps dans un tampon TNB dans un rapport de 1:100 et le conjugué HRP-α-biotine dans du tampon TNB, selon un rapport de 1:500. Retirez tout excès de tampon TNB les lames avec un mouchoir en papier. Lors de la détection deux transcrits simultanément, les deux anticorps peut aller à la fois. Appliquer un montant total de 100 ul de la solution d&#39;anticorps-TNB par puits et soigneusement couvrir avec une lamelle. Incuber les lames pendant 2 h. </li><li> Retirez les lamelles avec précaution. Laver les lames dans du tampon TNT 3 fois pendant 5 min. </li><li> Diluer le FITC-fluorophore dans le réactif d&#39;amplification tyramide à un ratio de 1:500 et appliquer 100 ul de la solution dans chaque puits. Couvrir les puits avec lamelles et laisser incuber pendant 12 min. </li><li> Retirez le couvercle glisse avec précaution et laver les lames 3 fois pendant 5 minutes dans le tampon TNT par agitation douce. </li><li> Diluer le Cyan3-fluorophore dans le réactif d&#39;amplification tyramide à un rapport de 1:500. Ajouter 100 ul paret de cette solution. Couvrir les puits avec lamelles et laisser incuber pendant 12 min. </li><li> Retirez le couvercle glisse soigneusement puis rincez rapidement les diapositives par immersion dans du tampon TNT. </li><li> Laver les lames 3 fois avec du tampon TNT pendant 5 min. </li><li> Immerger les lames rapidement en eau traitée au DEPC. </li><li> Laisser les lames sécher à l&#39;air. Monter les préparations anti-décoloration réactif contenant du DAPI. Sceller les coins des lamelles avec du vernis à ongles. </li><li> Les diapositives sont maintenant prêts pour la microscopie. Gardez les lames recouvertes d&#39;une feuille d&#39;aluminium et conserver à 4 ° C si l&#39;expérience doit être rempli le jour suivant. Une étanchéité complète des faces des lames peut être effectuée 24 heures après l&#39;application du réactif anti-décoloration. Cela permettra aux diapositives à numériser jusqu&#39;à une semaine après le protocole a été complété. </li></ol> 6. Visualisation des sondes hybridées <ol><li> Allumez le microscope. Un microscope standard immunofluorescence est sufficient pour ce genre d&#39;expérience, mais si vous avez accès à un microscope confocal, vous pouvez également l&#39;utiliser pour des images en 3D ou pour obtenir des vidéos de vos cellules colorées. Laissez microscope pour se réchauffer pendant 15-30 min. Allumez l&#39;ordinateur et le logiciel. </li><li> Vérifier la qualité de la coloration au microscope par immunofluorescence. Choisissez un endroit contenant quelques parasites. </li><li> Ajuster le gain de chaque laser manuellement. Ajuster le séjour de pixels à 4-6 m -1 -1 s et les étapes à 512 x 512 pixels. </li><li> Prenez une photo de test en utilisant Cadre Lambda. Réajuster le gain laser. </li><li> Prenez un minimum de 20 bonnes photos de fluorescence des cellules individuelles. Au moins 2-5 photos d&#39;un champ contenant 5-20 parasites sont nécessaires afin d&#39;obtenir une vue d&#39;ensemble juste le pourcentage des parasites positifs. </li></ol>

<ol>
	<li>Joergensen, L., Bengtsson, D. C., Bengtsson, A., Ronander, E., Berger, S. S., Turner, L., Dalgaard, M. B., Cham, G. K., Victor, M. E., Lavstsen, T., Theander, T. G., Arnot, D. E., Jensen, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+co-expression+of+two+different+PfEMP1+antigens+on+single+Plasmodium+falciparum-infected+erythrocytes+facilitates+binding+to+ICAM1+and+PECAM1.">Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1.</a> PLoS Pathog. 2, 1001083 (2010).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. 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+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat. Protoc. 3, 1370-1379 (2008).</li><li>Cheeseborough, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> District Laboratory Practice in Tropical Countries. , (2006).</li><li>Brolin, K. J., Ribacke, U., Nilsson, S., Ankarklev, J., Moll, K., Wahlgren, M., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+transcription+of+duplicated+var2csa+gene+copies+in+individual+Plasmodium+falciparum+parasites.">Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites.</a> Genome Biol. 10, R117 (2009).</li><li>Epp, C., Li, F., Howitt, C. A., Chookajorn, T., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+associated+sense+and+antisense+noncoding+RNAs+are+transcribed+from+the+var+gene+family+of+virulence+genes+of+the+malaria+parasite+Plasmodium+falciparum.">Chromatin associated sense and antisense noncoding RNAs are transcribed from the var gene family of virulence genes of the malaria parasite Plasmodium falciparum.</a> RNA. 15, 116-127 (2009).</li><li>Freitas-Junior, L. H., Hernandez-Rivas, R., Ralph, S. A., Montiel-Condado, D., Ruvalcaba-Salazar, O. K., Rojas-Meza, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+heterochromatin+propagation+and+histone+acetylation+control+mutually+exclusive+expression+of+antigenic+variation+genes+in+malaria+parasites.">Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites.</a> Cell. 121, 25-36 (2005).</li><li>Obernosterer, G., Martinez, J., Alenius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid-based+in+situ+detection+of+microRNAs+in+mouse+tissue+sections.">Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections.</a> Nature Protoc. 2, 1508-1514 (2007).</li><li>Li, F., Sonbuchner, L., Kyes, S. A., Epp, C., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+non-coding+RNA+are+transcribed+from+the+centromeres+of+Plasmodium+falciparum+and+are+associated+with+centromeric+chromatin.">Nuclear non-coding RNA are transcribed from the centromeres of Plasmodium falciparum and are associated with centromeric chromatin.</a> J. Biol. Chem. 283, 5692-5698 (2008).</li><li>Thompson, J., Sinden, R. E. <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+detection+of+Pbs21+mRNA+during+sexual+development+of+Plasmodium+berghei.">In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei.</a> Mol. Biochem. Parasitol. 68, 189-196 (1994).</li><li>Thompson, J. <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+detection+of+RNA+in+blood-+and+mosquito-stage+malaria+parasites.">In situ detection of RNA in blood- and mosquito-stage malaria parasites.</a> Methods Mole Med. 72, 225-235 (2002).</li><li>Arnot, D. E., Ronander, E., Bengtsson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progression+of+the+intra-erythrocytic+cell+cycle+of+Plasmodium+falciparum+and+the+role+of+the+centriolar+plaques+in+asynchronous+mitotic+division+during+schizogony.">The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony.</a> Int. J. Parasitol. 41, 71-80 (2011).</li><li>Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+genes+dynamically+colocalize+to+shared+sites+of+ongoing+transcription.">Active genes dynamically colocalize to shared sites of ongoing transcription.</a> Nat. Genet. 36, 1065-1071 (2004).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Analyse FISH ARN, contrairement aux méthodes telles que Northern blot et RT-PCR, permet la discrimination des transcrits d&#39;ARNm spécifiques au niveau de la cellule unique. Cela permet de faire la distinction entre les cellules transcriptionnellement actifs et inactifs, dans cet exemple, P. falciparum protozoaires parasites à l&#39;intérieur des globules rouges humains. Ces cellules entières observations sont souvent nécessaires et peuvent percer importantes et nouvelles tendances de la transcription <...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Acide acétique </td><td> Sigma / Aldrich </td><td> 338 826-100ml </td><td></td></tr><tr><td> Albumax médias: solution de RPMI Glutamine 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml de RPMI 1640 Une solution à 5 ml de glutamine </td></tr><tr><td> Albumax médias: Gentamicine sulfate Albumax solution </td><td> Lonza </td><td> BE02-012E </td><td> 2,5 ml de gentamycine sulfate 50 ml de solution Albumax </td></tr><tr><td> Albumax solution: Hypoxanthine </td><td> Sigma-Aldrich </td><td> H9377 </td><td> 0,8 g Hypoxanthine </td></tr><tr><td> Albumax solution: AlbuMAX II </td><td> Jenvitrogen </td><td> 11021-037 </td><td> 200 g Albumax </td></tr><tr><td> Albumax solution: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 4 litres du milieu RPMI 1640 Dissoudre avec aimant au max. 50 ° C. Filtrez stériliser et conserver à -20 ° C en aliquots. </td></tr><tr><td> Amberlite </td><td> Sigma </td><td> A5710-110G </td><td> Résine de deionize formamide. </td></tr><tr><td> Anti-biotine conjugué à la peroxydase de chèvre pAb </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> Anticorps anti-DIG </td><td> Novus biologiques </td><td> NB100-41330 </td><td></td></tr><tr><td> Anti-fade réactif avec DAPI </td><td> Invitrogen </td><td> P36931 </td><td> Les milieux de montage doit durcir pendant 24 heures avant de sceller complètement la diapositive. </td></tr><tr><td> Biotine ARN étiquetage Mix </td><td&gt; Roche </td><td> 11685597910 </td><td></td></tr><tr><td> Appareil photo numérique </td><td> Numérique Nikon F11 de vue CC </td><td></td><td></td></tr><tr><td> Lamelles de </td><td> Menzel-Glaser </td><td> 631-1570 </td><td></td></tr><tr><td> flacon de culture de 25 cm 2 de surface Nunclon </td><td> Nunc </td><td> 156340 </td><td></td></tr><tr><td> DEPC </td><td> Fluka / Sigma </td><td> 32490-100ml </td><td> 1 ml DEPC dans 1000 ml d&#39;eau deinonized. Ajouter un barreau aimanté et remuer pendant 12 h. Autoclave pendant 30 min. </td></tr><tr><td> Appareil photo numérique </td><td> Numérique Nikon F11 de vue CC </td><td></td><td></td></tr><tr><td> Protéines Dynabeads A </td><td> Invitrogen </td><td> 10002D </td><td></td></tr><tr><td> DynaMaq-15 Aimant </td><td> Invitrogen </td><td> 123-01D </td><td></td></tr><tr><td> Formamide bioultra 99% </td><td> Fluka/ Sigma </td><td> 47671-1l-F </td><td> Formamide désionisée: 5 g de résine échangeuse d&#39;ions par 100 ml de formamide. Mélanger 30 min. Filtrer sur papier Whatman. </td></tr><tr><td> . Gélatine 0 solution à 75%: gélatine </td><td> Sigma-Aldrich </td><td> G2500 </td><td> 3,75 gélatine g dans 500 ml de RPMI 1640. La chaleur à 56 ° C pour dissoudre. Stériliser par filtration alors que 56 ° C. Conserver à -20 ° C en aliquots. </td></tr><tr><td> . Gélatine 0 solution à 75%: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 3,75 gélatine g dans 500 ml de RPMI 1640. La chaleur à 56 ° C pour dissoudre. Stériliser par filtration alors que 56 ° C. Conserver à -20 ° C en aliquots. </td></tr><tr><td> Solution Glutamine: L-glutamine </td><td> Sigma-Aldrich </td><td> G3126 </td><td> 14,6 g L-glutamine dans 500 ml de NaCl 0,9%. Dissoudre, filtre sterilize et stocker à -20 ° C en aliquots. </td></tr><tr><td> Solution Glutamine: HCl </td><td> Sigma </td><td> H1758 </td><td> 14,6 g L-glutamine dans 500 ml de NaCl 0,9%. Dissoudre, filtrer stériliser et conserver à -20 ° C en aliquots. </td></tr><tr><td> Solution d&#39;hybridation: formamide Bio ultra 99% SSC 20x </td><td> Fluka / Sigma </td><td> 47671-1l-F </td><td> Total 20 ml, garder congelé à -20 ° C en aliquots 10 ml de formamide désionisée </td></tr><tr><td> Solution d&#39;hybridation: Concentré 50x Denhardt </td><td> Sigma </td><td> D2532 </td><td> 5ml 20xSSC </td></tr><tr><td> Solution d&#39;hybridation: Levure tRNARoche blocage réactif </td><td> Sigma </td><td> R-6750 </td><td> 2 ml 50x Denhardt 250 ul de 20 mg par ml de levure ARNt </td></tr><tr><td><td> Fluka / Sigma </td><td> 31149-106GF </td><td> 0.4g Roche réactif de blocage 1 ml de 10 mg par ml d&#39;ADN de sperme de saumon (Critique: saumon dénaturent spermDNA à 96 ° C pendant 5 min avant d&#39;ajouter à la solution d&#39;hybridation) </td></tr><tr><td> Hybridizer ThermoStar 100 HC4 </td><td> Quantifoil Instruments GmbH </td><td> 1004-0011 </td><td> Peut être remplacé par un four d&#39;hybridation et d&#39;hybridation RNase chambres libres rembourrés avec de l&#39;eau DEPC. </td></tr><tr><td> Immersion huile transparente aux UV fluorescence gratuit </td><td> Sigma </td><td> 10976-1EA </td><td></td></tr><tr><td> Microscope confocal immunofluorescence ou </td><td> Nikon D-Eclipse TE2000C </td><td></td><td></td></tr><tr><td> Vernis à ongles </td><td> Disponible dans n&#39;importe quelle pharmacie </td><td></td><td></td></tr><tr><td> PBS 20x: NaCl KCl Na 2 HPO 4 x 2H 2 O KH 2 PO 4 DEPC désionisée H 2 O </td><td></td><td></td><td> Ajuster le pH à 7,4 160 g 4 g 23 g 4 g 1 l </td></tr><tr><td> Paraformaldéhyde à 4% </td><td> Fluka / Chemika </td><td> 76240 </td><td> 4 g PFA dans 80 ml de PBS / DEPC. La chaleur à 65 ° C jusqu&#39;à ce que le PFA dissout. Ajouter 20 ml de PBS, laisser la solution refroidir. Ajuster le pH à 7,4. Filtrer. Boutique en aliquotes à -20 ° C. </td></tr><tr><td> Paraformaldéhyde à 4% / acide acétique à 5% </td><td></td><td></td><td> 950 paraformaldhyde ul + 50 ul d&#39;acide acétique </td></tr><tr><td> Pepsine </td><td> Sigma / Aldrich </td><td> P7000 </td><td></td></tr><tr><td> Conjugué à la peroxydase anti-biotine </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> RBC-milieu de lavage: solution de RPMI Glutamine 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml de RPMI 1640 une solution à 5 ml de glutamine </td></tr><tr><td> RBC-milieu de lavage: le sulfate de gentamicine </td><td> Lonza </td><td> BE02-012E </td><td> 2,5 ml de gentamycine sulfate </td></tr><tr><td> RNase </td><td> Sigma / Aldrich </td><td></td><td> Faire un 10 mg / ml de solution. </td></tr><tr><td> RNase tube de 1,5 ml </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> RNase Zap </td><td> Ambion </td><td> AM9780 </td><td></td></tr><tr><td> Diapositives 11mm 4 puits </td><td> Thermo Scientific </td><td> MENZXER306W </td><td></td></tr><tr><td> SSC 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> 3 M de NaCl (175 g / l) 0,3 M citrate de Na 3 x H 2 O (88 g / l) Ajuster à pH 7,0 avec HCl 1M </td></tr><tr><td> SSC 20x: citrate de sodium dihydraté </td><td> Sigma / aldrich </td><td> W302600 </td><td> 3 M de NaCl (175 g / l) 0,3 M citrate de Na 3 x H 2 O (88 g / l) Ajuster à pH 7,0 avec HCl 1M </td></tr><tr><td> PESS 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> 175,3 g de NaCl </td></tr><tr><td> PESS 20x: NaH 2 PO 4 </td><td> Sigma / Aldrich </td><td> S0751 </td><td> 27,6 g de NaH 2 PO 4. </td></tr><tr><td> PESS 20x: 4 EDTA en poudre </td><td></td><td></td><td> 9,4 g d&#39;EDTA en poudre Ajouter de l&#39;eau DEPC, ajuster le pH 7,4. Autofendit pendant 20 min </td></tr><tr><td> TNB mémoire tampon: tampon TNT </td><td></td><td></td><td> 10 ml du tampon TNT </td></tr><tr><td> TNB mémoire tampon: réactif de blocage </td><td></td><td></td><td> 0,05 g de réactif de blocage Pour dissoudre le réactif de blocage, chauffer la solution à 60 ° C pendant une heure sous agitation. Conserver à -20 ° C. (Dérivé de l&#39;Perkin Elmer TSA-protocole). </td></tr><tr><td> TNT mémoire tampon: Tris / HCl </td><td> Sigma </td><td> T1503 </td><td> 1M Tris / HCl, pH 8,0 </td></tr><tr><td> TNT mémoire tampon: NaCl </td><td> Sigma Aldrich </td><td> S9625 </td><td> 100 ml </td></tr><tr><td> Tampon TNT: Tween20 </td><td> Sigma </td><td> 93773 </td><td> 5 M NaCl 30 ml 1 ml DEPC dH 2 O869 Ml Ajuster le pH à 7,5 à la température ambiante. (Dérivé de l&#39;Perkin Elmer TSA-protocole). </td></tr><tr><td> TSA, plus Cyanine3 / fluorescéine système </td><td> Perkin Elmer </td><td> NEL753000IKT </td><td> Lire le protocole de la TSA Plus System Palette de fluorescence avec soin avant de commencer l&#39;expérience. </td></tr><tr><td> Tubes 14 ml stérile </td><td> Almeco - CM Aps LAB </td><td> 91016 </td><td></td></tr></tbody></table>

analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)

L&#39;adhérence des érythrocytes infectés par Plasmodium falciparum (IE) à l&#39;homme récepteurs endothéliaux au cours des infections de paludisme est médiée par l&#39;expression de PfEMP1 variantes de protéines codées par les gènes var. Le P. haploïde falciparum génome abrite environ 60 différents gènes var dont une seule a été soupçonnés d&#39;être transcrit par cellule à la fois pendant la phase de l&#39;infection du sang. Comment une telle réglementation mutuellement exclusif de la transcription des gènes var est atteint n&#39;est pas claire, ainsi que l&#39;identification des différents gènes var ou sous-groupes de gènes var associés aux différents récepteurs et les conséquences de la liaison différentielle sur les résultats cliniques de P. infections à P. falciparum. Récemment, le paradigme de la transcription mutuellement exclusive a été mise en doute par des essais de transcription individuelle sur la base de P. identification transcription falciparum chez inf uniqueète cellules érythrocytaires en utilisant l&#39;ARN d&#39;hybridation fluorescente in situ (FISH) de la transcription des gènes var par le parasite dans différents noyaux de P. falciparum IE 1. Ici, nous présentons un protocole détaillé pour la réalisation de la méthodologie ARN-FISH pour l&#39;analyse de la transcription des gènes var dans un seul noyau de P. falciparum infection des érythrocytes humains. La méthode est basée sur l&#39;utilisation de la digoxigénine marqué à la biotine et des sondes d&#39;ARN antisens en utilisant la TSA plus Fluorescence Palette Système 2 (Perkin Elmer), des analyses microscopiques et fraîchement sélectionné P. falciparum IE. Dans le procédé d&#39;hybridation in situ peut être utilisé pour contrôler la transcription et la régulation d&#39;une variété de gènes exprimés au cours des différentes étapes du P. cycle de vie falciparum et est adaptable à d&#39;autres espèces de parasites du paludisme et d&#39;autres organismes et des types cellulaires.

La figure 1 illustre un organigramme des principales étapes et pendant la durée de la méthodologie FISH ARN. Une série d&#39;images représentatives de bien et mal conservés tachées expériences FISH ARNm en utilisant unique P. falciparum IE sont présentés dans la figure 2. Ce protozoaire intracellulaire a une petite (1-1.5 um de diamètre) dans le noyau qui est coloré en bleu au DAPI. Vingt-quatre heures après l&#39;invasion érythrocytai...

Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

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

Fluorescent<em&gt; In situ</emHybridation&gt; (FISH) pour identifier des transcrits d&#39;ARNm dans des cellules individuelles permet l&#39;analyse de l&#39;activité polygénique tel que la transcription simultanée de plus d&#39;un membre de la<em&gt; Var</emFamille multigénique&gt; dans<em&gt; Plasmodium falciparum</em&gt; Érythrocytes infectés<sup&gt; 1</sup&gt;. La technique est adaptable et peut être utilisé sur différents types de gènes, des cellules et des organismes.

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 ...

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

15.3K vues.  University of Copenhagen. Fluorescent In situ (FISH) pour identifier des transcrits d'ARNm dans des cellules individuelles permet l'analyse de l'activité polygénique tel que la transcription simultanée de plus d'un membre de la Var dans Plasmodium falciparum Érythrocytes infectés 1. La technique est adaptable et peut être utilisé sur différents types de gènes, des cellules et des ...

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

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 ...

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Biologie

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 vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

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

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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 ...

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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

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

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse de Global ARN de synthèse à la Cellule Plain suivante hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...