Il lavoro è stato sostenuto in parte dal National Institute of Drug Abuse Center (P50 DA 011.806) e dell&#39;esercito degli Stati Uniti Dipartimento della Difesa (W81XWH-07-1-0183).

1. Stampa di microarray miRNA personalizzate <ol><li> Stampare il microarray diapositive utilizzando i servizi di base impianto di microarray presso un&#39;università o una società. La qualità dei microarray fabbricazione è uno dei fattori più critici per il successo di un esperimento di microarray. Provate alcuni servizi, se possibile. Idealmente, si potrebbe stampare 50-100 diapositive alla volta, e tutte le diapositive sarà identico, con ben separate, i punti individuali. </li><li> Per il supporto microarray, usiamo il II APERTURE vetrini rivestiti. </li><li> Per le sonde miRNA, si usa il nCode multispecie miRNA Microarray Probe Set V2. </li><li> Sciogliere tutti gli oligonucleotidi in 3 x SSC a 10 micron e quadruply li stampa nelle diapositive. Fissare gli scivoli per irraggiamento ultravioletto in base alle istruzioni per le diapositive II GAAP. Etichettare il vetrino con una penna di diamante per segnare il territorio e con le sonde lato stampato. Conservare a 22 ° C. </li></ol> 2. Preparazione del campione <ol><li> Qualsiasi metodoo reagente può essere utilizzato per isolare l&#39;RNA, fintanto che conserva piccoli RNA. Generalmente usiamo Trizol (Invitrogen) per estrarre l&#39;RNA totale, con quantità soddisfacente e la qualità delle preparazioni di RNA, come misurato da A a 260 nm e 280 nm A letture. </li><li> Per RNA etichettatura, mix ~ 25 mg di RNA totale con 0,5 mg di 5&#39;-PCU-DY547-3 &#39;6 in 1 tampone x (50 HEPES mM, pH 7.8, 20 mM MgCl 2, 10 mcg / ml BSA, il 10% DMSO) contenente ~ 20 unità T4 RNA ligasi 1, integrato con ~ 10 mM DTT e ~ 0,02-0,03 volume finale di x 10 T4 RNA ligasi 1 tampone per l&#39;ATP. </li><li> Consentono l&#39;uso di procedere sul ghiaccio all&#39;interno di un frigorifero per 2-24 ore. Precipitare l&#39;RNA con 0,3 M di acetato di sodio e 3 volumi di etanolo. Per la legatura efficiente e precipitazioni, dissolvere RNA totale di oltre 2 mg / ml, e mantenere il volume totale legatura a ~ 20 l. <ol><li> Se meno RNA è disponibile, la quantità di input RNA e 5&#39;-PCU-DY547-3 &#39;può essere ridimensionato. &lt;/ Li&gt; <li> Se l&#39;RNA è molto diluita, aggiungere vettore come il tRNA di lievito per aiutare nelle precipitazioni. </li></ol></li><li> Combinare ed etichetta in totale 1 mg di oligonucleotidi DNA di riferimento corrispondente a un sottoinsieme dei miRNA mammiferi utilizzando il Ulysis Alexa Fluor 647 Nucleic Acid Kit Etichettatura 6 come controllo per il processo di ibridazione. Purificare il DNA marcato con una colonna CentriSep e salvare nel buio a -20 ° C. </li></ol> 3. Microarray di ibridazione <ol><li> Per utilizzare le diapositive per la prima volta, pre-ibridare le diapositive in una soluzione filtrata di 3 x SSC, 0,1% SDS, 0.2% BSA per 30-60 minuti a 37 ° C. Immergere in acqua un paio di volte, poi in isopropanolo. Asciugare le diapositive utilizzando una centrifuga con un adattatore per diapositive a 100 g per 5 min a 22 ° C. </li></ol> Nota: Noi usiamo Corning camere di ibridazione microarray e Erie Scientific mSeries di Lifterslips per eseguire ibridazione microarray. <ol start="2"><li> Sciacquare il lifterslips in acqua, poi in etanolo, prima di asciugare in aria. Posizionare una diapositiva all&#39;interno di una base della camera di ibridazione microarray, e un lifterslip sulla parte superiore della diapositiva. Il lifterslip coprirà l&#39;area della sonda, con le sue strisce bianche contattando la diapositiva. </li><li> Nel buio, rallenta il precipitato, l&#39;RNA etichettati. Lavare una volta con etanolo al 70%, e l&#39;aria secca il pellet. Il pellet deve essere rossiccio. </li><li> Preparare una soluzione di ibridazione contenente 400 mM Na 2 HPO 4 pH 7,0, lo 0,8% di BSA, 5% SDS, 12% formammide 6 con 1/15-1/100 microlitri della purificato, marcato DNA di riferimento (2,4) per ogni campione di RNA. La quantità di DNA di riferimento aggiunto dipende da quanti oligonucleotidi diversi vengono etichettati (2,4). Se ci sono oltre 100, quindi meno di diluizione è necessario. </li><li> Sciogliere il pellet di RNA bene con ~ 60 microlitri della soluzione di ibridazione. </li><li> Aggiungere 20 ml di acqua per ciascuno dei pozzi umidificazione nella base ibridazione camera Corning microarray. </li><li> Aggiungi tsi miscela di RNA e DNA marcato riferimento alla diapositiva. Utilizzare una punta sottile pipetta, toccare delicatamente il bordo del lifterslip, e consentire la soluzione di inserire lo spazio tra le lifterslip e far scorrere mediante aspirazione. </li><li> Posizionare la camera di ibridazione coprono oltre la base, poi sigillare la camera con clip in metallo. </li><li> Mettete tutta la camera all&#39;interno del sacchetto di plastica che viene fornito con la cassetta camera da Corning. </li><li> Posizionare la cassetta camera (s) all&#39;interno di un contenitore con un tovagliolo di carta bagnata, coprire il recipiente con pellicola trasparente e metterla in un 37 ° C umidi, incubatore per colture cellulari ~ 24 ore. Queste precauzioni (3.6, 3.9, 3.10) garantire che la soluzione di ibridazione non si secchi durante l&#39;incubazione. </li></ol> 4. Post-ibridazione di elaborazione <ol><li> Smontare le cassette camera uno per uno. Immergere una diapositiva e la sua lifterslip in 2 x SSC a 22 ° C. Il lifterslip cadrà naturalmente dal vetrino. Luogo in 0,8 x SSC. Lavarele diapositive di 0,8 x SSC due volte, poi tre volte in 0,4 x SSC, 1-2 minuti in totale. Asciugare le diapositive utilizzando una centrifuga con un adattatore per diapositive. Lavare la lifterslips con acqua e risparmiare per il riutilizzo in seguito. </li><li> Scansione le diapositive con qualunque scanner adatto, ad esempio, la Perkin Elmer ScanArray 5000 o Molecular Devices Axon 4000B GenePix scanner microarray. Scansione a lunghezze d&#39;onda vicino a 547 nm e 647 nm per ottenere i file immagine corrispondente. </li><li> Utilizzare programmi come BlueFuse di quantificare le intensità dei pixel in ingresso, i file di immagine da 4,2). Ispezionare i singoli punti con il programma di escludere macchie anomale sul microarray da ogni ulteriore esame. Queste macchie anomale di solito nascono da stampa scivolare poveri o far scorrere la manipolazione dopo ibridazione. </li><li> Utilizzare programmi come GeneSpring ed Excel per l&#39;analisi dei dati e la presentazione. Considerazioni generali per la normalizzazione dei dati microarray applicare, che è oltre la portata di questo documento. </li><li> Una volta che i segnali soddisfacenti si ottengono after 4.3), striscia le diapositive microarray per il riutilizzo 7. Sciacquare i vetrini in acqua, poi immergere in pre-riscaldato, 1 mM NaOH e 0,1 x SSC in un piatto di colorazione a 62 ° C per 10-20 minuti. Ripetere l&#39;incubazione una volta. Lavare le diapositive un paio di volte in acqua per un massimo di 60 minuti agitando delicatamente a 22 ° C. Asciugare le diapositive utilizzando una centrifuga, e conservare a 22 ° C. </li><li> Per utilizzare le diapositive rigenerata, non è necessario per loro pre-ibridare di nuovo. Basta lavarli in acqua seguito da isopropanolo, e asciugare le diapositive a destra prima di ibridazione. </li></ol> 5. Rappresentante dei risultati: Abbiamo in gran parte seguite le procedure descritte al profilo di espressione globale miRNA in migliaia di campioni, cioè RNA isolato dal pesce zebra a campione umano in diverse condizioni. La figura 1 mostra una immagine digitalizzata di un microarray per dimostrare segnali di ibridazione molto precisa e forte sul diapositive. La Pearson correlatisui coefficienti tecnici tra repliche di ibridazione microarray sono ~ 0.99 7, indicando eccellente riproducibilità. <img alt="Figura 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> Figura 1 l&#39;immagine composita di una diapositiva scansione microarray miRNA dopo ibridazione. Macchie rosse il risultato di ibridazioni dal DNA di riferimento, le macchie verdi dal DY547 marcato campione di RNA, mentre i punti gialli erano di ibridazioni sia dal DNA e RNA per la stessa sonda.

<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>

Nessun conflitto di interessi dichiarati.

Nonostante i recenti progressi nelle tecnologie di sequenziamento profondo, microarray rimane una scelta praticabile per high-throughput analisi del DNA e RNA. Rispetto al sequenziamento profondo, gli esperimenti microarray sono più economici, e un laboratorio di biologia molecolare tipico in grado di eseguire la maggior parte degli esperimenti e analisi dei dati in-house, che consente flessibilità e consente di risparmiare tempo. In futuro, microarray probabilmente adatto a interrogare intensamente insiemi di geni, p...

<table border="1"><tbody><tr><th> Articoli </th><th> Venditore </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> NCode multi-specie miRNA Microarray Probe Set V2 </td><td> Invitrogen </td><td> MIRMPS201 </td><td> Progettato sulla base del miRBase versione 9.0 (ottobre 2006). Esso contiene ~ 1140 non modificato, 34-44 oligonucleotidi nt lungo come sonde per verme, volare, zebrafish, topo, ratto, e miRNA umani, e una serie di sonde di controllo interno, come snoRNAs. Le sonde miRNA sono doppietti delle sequenze complementari a miRNA maturo, quindi le dimensioni di ~ 44 nt. Per l&#39;analisi si può concentrare su miRNA da un genoma particolare (s) di interesse. </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596018 </td><td> Abbiamo usato anche arricchito, piccola frazione di RNA per l&#39;etichettatura, anche se campioni di RNA totale sono più veloci e più facili da preparare e di quantificare e adatto per applicazioni a valle quali l&#39;analisi di mRNA. </td></tr><tr><td> T4 RNA ligasi 1 </td><td> New England Biolabs </td><td> M0204L </td><td></td></tr><tr><td> Ulysis Alexa Fluor 647 Nucleic Acid Kit Etichettatura </td><td> Invitrogen </td><td> U21660 </td><td> Questo kit o prodotti simili possono essere usati per l&#39;etichetta sperimentale campioni di RNA o RNA di controllo (invece di DNA di controllo) pure. </td></tr><tr><td> 5&#39;-PCU-DY547-3 &#39; </td><td> Dharmacon </td><td> Una soluzione su misura </td><td> Piccola frazione di RNA possono essere etichettati in modo simile da legatura. </td></tr><tr><td> CentriSep colonne </td><td> Princeton separazioni </td><td> CS-901 </td><td></td></tr><tr><td> GAPSII scivolo rivestito </td><td> Corning </td><td> 40004 </td><td> Altri tipi di diapositive può essere utilizzato anche. </td></tr><tr><td> Microarray ibridazione camere </td><td> Corning </td><td> 2551 o 40080 </td><td> Altri tipi di camere di ibridazione e coprioggetto dovrebbe funzionare. Utilizzo di macchinari ibridazione disponibili in commercio possono ridurre il tempo di ibridazione in modo significativo, ad esempio, per circa 2 ore. </td></tr><tr><td> Lifterslips </td><td> ThErmo / Erie scientifico </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

microRNA (miRNA) sono una grande famiglia di circa 22 nucleotidi (nt) lungo molecole di RNA che sono ampiamente espresse in eucarioti 1. Genomi complessi codificare almeno centinaia di miRNA, che in primo luogo inibire l&#39;espressione di un vasto numero di geni bersaglio post-trascrizionalmente 2, 3. miRNA controllare una vasta gamma di processi biologici 1. Inoltre, l&#39;espressione di miRNA alterata è stata associata a malattie umane come i tumori, e miRNA possono servire come biomarcatori per le malattie e la prognosi 4, 5. E &#39;importante, quindi, per capire l&#39;espressione e le funzioni dei miRNA in diverse condizioni. Tre approcci principali sono stati impiegati per profilo di espressione di miRNA: real-time PCR, microarray e sequenziamento profondo. La tecnica dei microarray miRNA ha il vantaggio di essere high-throughput, generalmente meno costose, e la maggior parte delle fasi sperimentali e di analisi può essere carrIED in un laboratorio di biologia molecolare alla maggior parte delle università, scuole di medicina e gli ospedali associati. Qui, descriviamo un metodo per l&#39;esecuzione di esperimenti microarray su ordinazione miRNA. Un insieme sonda miRNA verrà stampato su vetrini microarray per produrre miRNA. RNA è isolato utilizzando un metodo o reagente che conserva le piccole specie di RNA, e poi etichettato con un colorante fluorescente. Come controllo, oligonucleotidi DNA di riferimento corrispondente a un sottoinsieme dei miRNA sono etichettati con un colorante diverso fluorescenza. Il DNA di riferimento servirà per dimostrare la qualità della diapositiva e l&#39;ibridazione e sarà anche utilizzata per la normalizzazione dei dati. L&#39;RNA e DNA si mescolano e ibridato a una diapositiva microarray contenenti sonde per la maggior parte dei miRNA nel database. Dopo il lavaggio, la diapositiva viene sottoposto a scansione per ottenere immagini e intensità dei punti individuali quantificati. Questi segnali prime saranno ulteriormente elaborati e analizzati i dati di espressione dei miRNA corrispondente. MicroArray diapositive possono essere rimossi e rigenerata per ridurre i costi di microarray e di migliorare la coerenza degli esperimenti microarray. Gli stessi principi e le procedure sono applicabili ad altri tipi di esperimenti microarray su ordinazione.

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

Performing Custom MicroRNA Microarray Experiments

Una semplice procedura di eseguire esperimenti microarray su ordinazione microRNA è descritto. I passaggi includono isolare RNA, l&#39;etichettatura RNA e DNA di riferimento, ibridando i campioni di microarray, la scansione del microarray, quantificare e analizzare segnali di ibridazione.

Esecuzione personalizzati Esperimenti di Microarray 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 Views.  University of Minnesota. Una semplice procedura di eseguire esperimenti microarray su ordinazione microRNA è descritto. I passaggi includono isolare RNA, l'etichettatura RNA e DNA di riferimento, ibridando i campioni di microarray, la scansione del microarray, quantificare e analizzare segnali di ibridazione.

Video: 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 ascospore di produzione e di raccolta di esperimenti di microarray.

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

Ricerca

Biologia

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.

Preparazione di doppi strati artificiali per esperimenti Elettrofisiologia

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.

L&#39;elaborazione del Pino Loblolly 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.

Analisi di espressione genica globale Utilizzando un Zebrafish piattaforma Microarray oligonucleotidi

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.

Microarray analisi per 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.

Genome Editing con nucleasi zinco Finger CompoZr personalizzati (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; Ibridazione per tessuti renali fissati in formalina

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 consegna di una cuore specifici MicroRNA-spugna

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.

Strumento di editing genetico CRISPR per l'analisi della rete di cluster microRNA

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