Le travail a été soutenu en partie par le National Institute of Drug Abuse Centre (P50 DA 011 806) et United States Army Department of Defense (W81XWH-07-1-0183).

1. Impression de puces personnalisées miARN <ol><li> Imprimer la puce diapositives en utilisant les services biopuces installation de base à une université ou une entreprise. La qualité de fabrication de puces à ADN est l&#39;un des facteurs les plus critiques pour le succès d&#39;une expérience microarray. Essayez un peu de services si possible. Idéalement, il faudrait imprimer de 50 à 100 lames à la fois, et toutes les diapositives sera identique, avec bien séparés, les taches individuelles. </li><li> Pour le support de microréseau, nous utilisons des lames GAPS II couché. </li><li> Pour les sondes miARN, nous utilisons le nCode multi-espèces miARN Microarray Probe Set V2. </li><li> Dissoudre tous les oligonucléotides dans 3 x SSC à 10 uM et quadruply les imprimer sur les diapositives. Fixer les lames par irradiation aux ultraviolets conformément aux instructions pour les diapositives II GAAP. Étiquette de la diapositive avec un stylo de diamants pour marquer la zone et le côté avec les sondes imprimé. Magasin à 22 ° C. </li></ol> 2. La préparation des échantillons <ol><li> Toute méthodeou réactif peut être utilisé pour isoler l&#39;ARN, tant qu&#39;elle préserve les petits ARN. Nous utilisons généralement Trizol (Invitrogen) pour extraire l&#39;ARN total, avec la quantité satisfaisante et la qualité des préparations d&#39;ARN, tel que mesuré par A et A 260 nm lectures 280nm. </li><li> Pour l&#39;ARN étiquetage, mélanger ~ 25 ug d&#39;ARN total avec 0,5 ug de 5&#39;-PCU-DY547-3 &#39;6 en 1 x tampon (50 mM HEPES, pH 7,8, 20 mM MgCl2, 10 ug / ml de BSA, 10% DMSO) contenant environ 20 unités de T4 ARN ligase 1, complétée par la TNT ~ ~ 10 mM et 0,02 à 0,03 volume final de la 10 x T4 ARN ligase 1 tampon pour l&#39;ATP. </li><li> Autoriser l&#39;étiquetage de procéder sur glace à l&#39;intérieur d&#39;un réfrigérateur pour les 2-24 heures. Précipiter l&#39;ARN, avec 0,3 M d&#39;acétate de sodium et 3 volumes d&#39;éthanol. Pour la ligature efficace et précipitations, dissoudre l&#39;ARN total à plus de 2 mg / ml, et maintenir le volume total de la ligature à ~ 20 pl. <ol><li> Si moins d&#39;ARN est disponible, le montant de l&#39;apport de l&#39;ARN et le 5&#39;-PCU-DY547-3 &#39;peuvent être revus à la baisse. &lt;/ Li&gt; <li> Si l&#39;ARN est très dilué, ajouter transporteur comme ARNt de levure pour l&#39;aide dans les précipitations. </li></ol></li><li> Combinez et étiquettes au total 1 ug d&#39;oligonucléotides d&#39;ADN de référence correspondant à un sous-ensemble des miARN de mammifères en utilisant les Ulysis Alexa Fluor 647 Kit Nucleic Acid Étiquetage 6 comme un contrôle pour le processus d&#39;hybridation. Purifier l&#39;ADN marqué à l&#39;aide d&#39;une colonne Centrisep et sauvegarder dans l&#39;obscurité à -20 ° C. </li></ol> 3. L&#39;hybridation microarray <ol><li> Pour utiliser les diapositives pour la première fois, la pré-hybridation des lames dans une solution filtrée de 3 x SSC, SDS 0,1%, 0,2% de BSA pendant 30-60 min à 37 ° C. Les submerger dans l&#39;eau à quelques reprises, puis à l&#39;isopropanol. Sécher les lames à l&#39;aide d&#39;une centrifugeuse avec un adaptateur diapositives à 100 g pendant 5 min à 22 ° C. </li></ol> Note: Nous utilisons des chambres Corning hybridation microarray et mSeries Érié scientifique de Lifterslips pour effectuer l&#39;hybridation de biopuces. <ol start="2"><li> Rincez le lifterslips dans l&#39;eau, puis dans de l&#39;éthanol, avant le séchage à l&#39;air. Placez une diapositive dans une base de microréseaux chambre d&#39;hybridation, et un lifterslip sur le dessus de la glissière. Le lifterslip couvrira la zone de la sonde, avec ses bandes blanches contactant la diapositive. </li><li> Dans l&#39;obscurité, le ralentissement de la précipitation, l&#39;ARN étiquetés. Lavez une fois avec 70% d&#39;éthanol, et l&#39;air sec du culot. Le culot doit être rougeâtre. </li><li> Préparer une solution d&#39;hybridation contenant 400 mM de Na 2 HPO 4 pH 7,0, 0,8% de BSA, 5% de SDS, le formamide 12% 6 avec 1/15-1/100 pl de la purifier, étiquetées ADN de référence (2,4) par échantillon d&#39;ARN. La quantité d&#39;ADN de référence ajoutée dépend du nombre de différents oligonucléotides sont étiquetés (2,4). S&#39;il ya plus de 100, alors moins de dilution est nécessaire. </li><li> Dissoudre le culot d&#39;ARN bien avec ~ 60 pl de la solution d&#39;hybridation. </li><li> Ajouter 20 ul d&#39;eau pour chacun des puits d&#39;humidification dans la base de microréseaux chambre d&#39;hybridation Corning. </li><li> Ajouter tIl mélange de l&#39;ARN et l&#39;ADN de référence étiqueté à la diapositive. Utiliser une pipette fine, toucher doucement le bord de la lifterslip, et permettre à la solution d&#39;entrer dans l&#39;espace entre les lifterslip et glisser à travers aspiration. </li><li> Placer la chambre d&#39;hybridation couvrent plus de la base, puis sceller la chambre avec des pinces métalliques. </li><li> Mettez toute la chambre à l&#39;intérieur du sac en plastique qui vient avec la cassette de chambre de Corning. </li><li> Placer la cassette de chambre (s) à l&#39;intérieur d&#39;un conteneur avec une serviette de papier mouillé; couvrez le récipient avec une pellicule plastique et placez-le dans un 37 ° C et humide, de culture cellulaire incubateur pour ~ 24 heures. Ces précautions (3,6, 3,9, 3,10) s&#39;assurer que la solution d&#39;hybridation ne se dessèche pas pendant l&#39;incubation. </li></ol> 4. Post-traitement de l&#39;hybridation <ol><li> Démonter les cassettes de chambre, un par un. Plonger une lame et son lifterslip en 2 x SSC à 22 ° C. Le lifterslip va naturellement tomber la lame. Placez-le dans 0,8 x SSC. Laverles diapositives de 0,8 x SSC deux fois, puis trois fois en 0,4 x SSC, 1-2 min au total. Sécher les lames à l&#39;aide d&#39;une centrifugeuse avec un adaptateur de diapositives. Laver les lifterslips avec de l&#39;eau et à économiser pour les réutiliser plus tard. </li><li> Numérisez les diapositives avec n&#39;importe quel scanner approprié, par exemple, l&#39;Perkin Elmer ScanArray 5000 ou Molecular Devices Axon GenePix 4000B biopuces scanner. Balayage des longueurs d&#39;onde proche de 547 nm et 647 nm pour obtenir les fichiers d&#39;image correspondante. </li><li> Utiliser des programmes tels que BlueFuse de quantifier les intensités des pixels dans l&#39;entrée, les fichiers image de 4,2). Inspectez taches individuelles avec le programme d&#39;exclure des taches anormales sur les microarrays d&#39;un examen plus approfondi. Ces taches anormales résultent habituellement d&#39;imprimerie glisser mauvaise manipulation ou une diapositive après hybridation. </li><li> Utiliser des programmes tels que GeneSpring et Excel pour l&#39;analyse des données et la présentation. Considérations générales pour la normalisation des données biopuces s&#39;appliquent, ce qui est au-delà de la portée du présent document. </li><li> Une fois les signaux satisfaisants sont obtenus after 4,3), la bande des lames microarray pour réutilisation 7. Rincer les lames dans de l&#39;eau, puis plonger dans préchauffé, NaOH 1 mm et 0,1 x SSC dans un plat à coloration à 62 ° C pendant 10-20 minutes. Répétez l&#39;incubation fois. Lavez le C. diapositives à quelques reprises dans l&#39;eau pendant 60 minutes avec agitation douce à 22 ° Sécher les lames à l&#39;aide d&#39;une centrifugeuse, et conserver à 22 ° C. </li><li> Pour utiliser les diapositives régénéré, il n&#39;est pas nécessaire de les pré-s&#39;hybrider à nouveau. Il suffit de les laver à l&#39;eau suivie de l&#39;isopropanol, et sécher les diapositives à droite avant l&#39;hybridation. </li></ol> 5. Les résultats représentatifs: Nous avons largement suivi les procédures décrites au profil de l&#39;expression du miARN mondial dans des milliers d&#39;échantillons, c&#39;est-à ARN isolé de poisson zèbre au spécimen humain dans diverses conditions. La figure 1 montre une image numérisée d&#39;une biopuce à démontrer signaux d&#39;hybridation très précis et très forte sur le diapositive. Le correlati Pearsonsur les coefficients techniques entre les répliques de l&#39;hybridation microarray sont ~ 0.99 7, indiquant une excellente reproductibilité. <img alt="Figure 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> Figure 1 Image composite d&#39;une lame de biopuces miARN numérisée après l&#39;hybridation. Taches rouges résulté de hybridations par l&#39;ADN de référence, des taches vertes de l&#39;échantillon DY547-ARN marqué, tandis que les taches jaunes étaient d&#39;hybridations à la fois par l&#39;ADN et d&#39;ARN pour les mêmes sondes.

<ol>
	<li>Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+animal+microRNAs.">The functions of animal microRNAs.</a> Nature. 431, 350-355 (2004).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Chekulaeva, M., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+miRNA-mediated+post-transcriptional+regulation+in+animal+cells.">Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.</a> Curr. Opin. Cell Biol. 21, 452-460 (2009).</li><li>Farazi, T. A., Spitzer, J. I., Morozov, P., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+human+cancer.">miRNAs in human cancer.</a> J. Pathol. 223, 102-115 (2011).</li><li>Small, E. M., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+roles+of+microRNAs+in+cardiovascular+biology.">Pervasive roles of microRNAs in cardiovascular biology.</a> Nature. 469, 336-342 (2011).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. 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+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat. Methods. 1, 47-53 (2004).</li><li>Zhang, X., Xu, W., Tan, J., Zeng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripping+custom+microRNA+microarrays+and+the+lessons+learned+about+probe:slide+interactions.">Stripping custom microRNA microarrays and the lessons learned about probe:slide interactions.</a> Anal. Biochem. 386, 222-227 (2009).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucl. Acids. Res. 36, D154-D158 (2008).</li><li>Landgraf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+microRNA+expression+atlas+based+on+small+RNA+library+sequencing.">A mammalian microRNA expression atlas based on small RNA library sequencing.</a> Cell. 129, 1401-1414 (2007).</li><li>Chiang, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs:+experimental+evaluation+of+novel+and+previously+annotated+genes.">Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.</a> Genes. Dev. 24, 992-1009 (2010).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Malgré les progrès récents dans les technologies de séquençage profond, biopuces reste un choix viable pour analyse à haut débit de l&#39;ADN et l&#39;ARN. Comparé à un séquençage en profondeur, les expériences biopuces sont moins chers, et un laboratoire de biologie moléculaire typique peut exécuter la plupart des expériences et l&#39;analyse des données en interne, ce qui permet une flexibilité et un gain de temps. Dans l&#39;avenir, les puces sont susceptibles bien adapté à interroger de manière i...

<table border="1"><tbody><tr><th> Articles </th><th> Vendeur </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> NCode multi-espèces miARN Microarray Probe Set V2 </td><td> Invitrogen </td><td> MIRMPS201 </td><td> Conçu sur la base des miRBase 9.0 Release (Octobre 2006). Il contient ~ 1140 non modifiées, des oligonucléotides de 34 à 44 nt longtemps que les sondes pour le ver, la mouche, le poisson zèbre, souris, rat, et miARN humains, et un certain nombre de sondes de contrôle interne tels que snoRNAs. Les sondes sont des doublets miARN des séquences complémentaires à miARN mature, donc la taille de ~ 44 nt. Pour l&#39;analyse, on peut se concentrer sur miARN à partir d&#39;un génome particulier (s) d&#39;intérêt. </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596018 </td><td> Nous avons également utilisé enrichi, petite fraction ARN pour l&#39;étiquetage, bien que les échantillons d&#39;ARN totaux sont plus rapides et plus faciles à préparer et à quantifier et adapté aux applications en aval tels que l&#39;analyse d&#39;ARNm. </td></tr><tr><td> T4 ARN ligase 1 </td><td> La Nouvelle-Angleterre Biolabs </td><td> M0204L </td><td></td></tr><tr><td> Ulysis Alexa Fluor 647 Kit Nucleic Acid Etiquetage </td><td> Invitrogen </td><td> U21660 </td><td> Ce kit ou des produits similaires peuvent être utilisés pour étiqueter les échantillons d&#39;ARN expérimentale ou d&#39;un ARN de contrôle (au lieu de l&#39;ADN de contrôle) ainsi. </td></tr><tr><td> 5&#39;-PCU-DY547-3 &#39; </td><td> Dharmacon </td><td> Custom made </td><td> Petite fraction d&#39;ARN peuvent être également étiquetés par ligature. </td></tr><tr><td> Colonnes Centrisep </td><td> Séparations Princeton </td><td> CS-901 </td><td></td></tr><tr><td> GAPSII lame revêtue </td><td> Corning </td><td> 40004 </td><td> D&#39;autres types de lames peuvent être également utilisés. </td></tr><tr><td> Chambres d&#39;hybridation microarray </td><td> Corning </td><td> 2551 ou 40080 </td><td> D&#39;autres types de chambres d&#39;hybridation et de lamelles devrait également fonctionner. En utilisant l&#39;hybridation des machines disponibles dans le commerce peut réduire significativement le temps l&#39;hybridation, par exemple, à 2 heures. </td></tr><tr><td> Lifterslips </td><td> Th.Ermo / Erie Scientific </td><td> 25X60I-M5439-001-LS </td><td></td></tr><tr><td> BlueFuse </td><td> BlueGenome </td><td></td><td></td></tr><tr><td> GeneSpring </td><td> Agilent </td><td></td><td></td></tr></tbody></table>

performing custom microrna microarray experiments

microARN (miARN) sont une grande famille de ~ 22 nucléotides (nt) de longues molécules d&#39;ARN qui sont largement exprimés dans les eucaryotes 1. Génomes complexes codent pour au moins plusieurs centaines de miARN, qui sont principalement inhiber l&#39;expression d&#39;un grand nombre de gènes cibles de post-transcriptionnel 2, 3. miARN contrôler un large éventail de processus biologiques 1. En outre, l&#39;expression du miARN modifié a été associée à des maladies humaines telles que les cancers, et miARN peuvent servir de biomarqueurs pour les maladies et le pronostic 4, 5. Il est donc important de comprendre l&#39;expression et les fonctions des miARN dans diverses conditions. Trois grandes approches ont été employées pour l&#39;expression du miARN profil: PCR en temps réel, puces à ADN et le séquençage en profondeur. La technique des biopuces miARN a l&#39;avantage d&#39;être à haut débit, généralement moins coûteux, et la plupart des étapes expérimentales et l&#39;analyse peuvent être CarrIED dans un laboratoire de biologie moléculaire à la plupart des universités, écoles de médecine et des hôpitaux associés. Ici, nous décrivons une méthode pour effectuer des expériences de micropuces personnalisées miARN. Un ensemble de sondes miARN seront imprimés sur des lames de verre pour produire des puces miARN. L&#39;ARN est isolé en utilisant une méthode ou d&#39;un réactif qui préserve les espèces de petits ARN, puis marquées avec un colorant fluorescent. Comme contrôle, oligonucléotides d&#39;ADN de référence correspondant à un sous-ensemble des miARN sont également marqués par un colorant différent de fluorescence. L&#39;ADN de référence servira à démontrer la qualité de la lame et l&#39;hybridation et sera également utilisé pour la normalisation des données. L&#39;ARN et l&#39;ADN sont mélangés et hybridés sur une lame de biopuces contenant des sondes pour la plupart des miARN dans la base de données. Après le lavage, la lame est scanné afin d&#39;obtenir des images et des intensités des spots individuels quantifiés. Ces signaux bruts seront encore traitées et analysées que les données d&#39;expression de l&#39;miARN correspondants. MicroAdiapositives rray peut être dépouillé et régénérée pour réduire le coût des puces à ADN et à renforcer la cohérence des expériences sur biopuces. Les mêmes principes et les procédures sont applicables à d&#39;autres types d&#39;expériences de micropuces personnalisées.

Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

Performing Custom MicroRNA Microarray Experiments

Une procédure simple d&#39;effectuer des expériences de micropuces personnalisées microARN est décrite. Les étapes comprennent isoler l&#39;ARN, l&#39;étiquetage ARN et l&#39;ADN de référence, l&#39;hybridation des échantillons à puces, la numérisation des microarrays, quantifier et analyser les signaux d&#39;hybridation.

Exécution personnalisée expériences de micropuces MicroRNA

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

performing-custom-microrna-microarray-experiments

Research

JoVE Journal

Biology

19.4K vues.  University of Minnesota. Une procédure simple d'effectuer des expériences de micropuces personnalisées microARN est décrite. Les étapes comprennent isoler l'ARN, l'étiquetage ARN et l'ADN de référence, l'hybridation des échantillons à puces, la numérisation des microarrays, quantifier et analyser les signaux d'hybridation.

Vidéo: Performing Custom MicroRNA Microarray Experiments

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae ascospores production et la collecte d&#39;expériences sur biopuces.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Recherche

Biologie

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Préparation de bicouches artificielle pour des expériences d&#39;électrophysiologie

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

Traitement du pin taeda PtGen2 cDNA microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Mondial Analyse expression génique utilisant une plateforme biopuces oligonucléotidiques Zebrafish

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Analyse de Microarray pour Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Génome édition avec des nucléases personnalisés CompoZr doigt de zinc (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

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

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

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

In Vivo Nanovector livraison d’une coeur spécifiques microARN-éponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

Outil d’édition de gènes CRISPR pour l’analyse de réseaux de microARN

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

Performing Muscle Cryoinjury on a Zebrafish

For performing the myomere cryoinjury on zebrafish, use a custom manufactured stainless steel cryoprobe. To avoid frostbite while handling the probe during the procedure, insert the probe handle into a pipette tip and secure it by wrapping it with tape. Before beginning with the cryoinjury procedure, prepare a beaker for the anesthesia working solution, a spoon for handling the fish, a moist sponge, and a tank with system water for post-procedure recovery of the fish.Prepare the anesthesia stock solution following the steps shown on the screen. Then add four milliliters of the prepared stock solution to 100 milliliters of system water in a beaker to prepare the tricaine working solution. To begin the cryoinjury procedure, immerse the probe in liquid nitrogen for a minimum of three minutes.Wet the sponge in system water and place it on a flat surface. Then one to two minutes after transferring a single adult zebrafish into the tricaine working solution, confirm its unresponsiveness by touching the fish gently with the spoon. Place the anesthetized fish onto the wet sponge.Locate the caudal peduncle posterior to the anal fin and anterior to the caudal fin. At this stage, remove the cryoprobe from the liquid nitrogen. Shake the probe gently to ensure no residual liquid nitrogen remains on the tip.Place the edge of the spatula perpendicular to the body on the caudal peduncle. Keep the probe in this position for six seconds without applying pressure, before releasing the cryoprobe from the tissue. Then transfer the fish into the system water in the recovery tank.Monitor the fish while it resumes breathing and swimming after waking up from the anesthesia. If opercular movements do not occur after 30 seconds, stimulate the fish by pipetting system water into the gills until the animal initiates respiration by itself. Video recording of control and cryoinjured fish at one day post-injury or DPCI revealed that although the cryoinjured fish swam less actively, they did not display any abnormal movements, such as swirling, convolution, or reduced equilibrium.In the husbandry system, their position in the tank and food intake was similar to those of the uninjured fish. As observed at 5 DPCI, normal behavior was still persistent indicating the cryoinjury procedure did not severely affect the wellbeing of the animals.