Die Arbeit wurde teilweise durch National Institute of Drug Abuse Center (P50 DA 011806) und United States Army Department of Defense (W81XWH-07-1 bis 0183) unterstützt.

1. Drucken von benutzerdefinierten miRNA-Mikroarrays <ol><li> Drucken Sie die Microarray-Slides mit Hilfe der Microarray-core facility services an einer Universität oder eines Unternehmens. Die Qualität der Microarray-Herstellung ist einer der wichtigsten Faktoren für den Erfolg eines Mikroarray-Experiment. Probieren Sie ein paar Dienste, wenn möglich. Idealerweise würde man 50-100 Dias zu einem Zeitpunkt, zu drucken und alle Folien werden identisch aussehen, mit gut voneinander getrennt, die einzelnen Spots. </li><li> Für Microarray-Unterstützung, verwenden wir die GAPS II beschichtete Objektträger. </li><li> Für miRNA Sonden, verwenden wir die NCode Multi-Species miRNA Microarray Probe Set V2. </li><li> Lösen Sie alle Oligonukleotide in 3 x SSC bei 10 uM und vierfach drucken sie auf den Folien. Befestigen Sie die Folien durch UV-Bestrahlung nach den Anweisungen für die GAAPS II rutscht. Beschriften Sie die Folie mit einem Diamant-Stift, um die Gegend und die Seite der gedruckten Sonden zu markieren. Lagerung bei 22 ° C. </li></ol> 2. Probenvorbereitung <ol><li> Jede Methodeoder Reagenz kann verwendet werden, um RNA zu isolieren, solange es kleine RNAs bewahrt. Wir verwenden in der Regel Trizol (Invitrogen) zur Gesamt-RNA-Extrakt, mit befriedigender Qualität und Quantität der RNA-Präparationen, wie von A 260nm und 280nm A gemessene. </li><li> Für die RNA-Kennzeichnung, Mix ~ 25 ug Gesamt-RNA mit 0,5 ug 5&#39;-PCU-DY547-3 &#39;6 in 1 x Puffer (50 mM HEPES, pH 7,8, 20 mM MgCl 2, 10 pg / ml BSA, 10% DMSO) mit ~ 20 Einheiten T4-RNA-Ligase 1, mit ~ 10 mM DTT und ~ 0,02-0,03 Endvolumen des 10 x T4-RNA-Ligase 1 Puffer für ATP ergänzt. </li><li> Lassen Sie die Kennzeichnung auf Eis im Kühlschrank für 2-24 Stunden ablaufen. Niederschlag RNA mit 0,3 M Natriumacetat und 3 Volumen Ethanol. Für eine effiziente Ligation und Niederschlag, löst Gesamt-RNA von über 2 mg / ml, und halten Sie die gesamte Ligation Volumen bei ~ 20 pl. <ol><li> Wenn weniger RNA ist verfügbar, die Menge der Eingangs-RNA und 5&#39;-PCU-DY547-3 &#39;kann verkleinert werden. &lt;/ Li&gt; <li> Wenn die RNA sehr verdünnt ist, fügen Träger, wie Hefe-tRNA an der Niederschläge zu unterstützen. </li></ol></li><li> Kombinieren und Etikett in insgesamt 1 pg von DNA-Oligonukleotiden entspricht einer Teilmenge von Säugetieren miRNAs mit dem Ulysis Alexa Fluor 647 Nucleic Acid Labeling Kit 6 als Kontrolle für die Hybridisierung Prozess. Purify der markierten DNA mit einem Centrisep Spalte und speichern in Dunkeln bei -20 ° C. </li></ol> 3. Microarray-Hybridisierung <ol><li> So verwenden Sie die Folien zum ersten Mal, vor hybridisieren die Folien in einer gefilterten Lösung von 3 x SSC, 0,1% SDS, 0,2% BSA für 30-60 min bei 37 ° C. Tauchen sie in Wasser ein paar Mal, dann in Isopropanol. Trocknen Sie die Folien mit einer Zentrifuge mit einem Dia-Adapter bei 100 g für 5 min bei 22 ° C. </li></ol> Hinweis: Wir verwenden Corning Microarray-Hybridisierung Kammern und Erie Scientific M-Serie von LifterSlips auf Microarray-Hybridisierung durchzuführen. <ol start="2"><li> Spülen Sie den lifterslips in Wasser, dann in Ethanol, vor dem Trocknen an der Luft. Setzen Sie ein Bild in einem Microarray-Hybridisierung Kammer Basis und eine lifterslip am oberen Rand der Folie. Die lifterslip deckt die Sonde Bereich, mit seinen weißen Streifen Kontaktierung der Folie. </li><li> In den dunklen, Spin-down der ausgefallene, markierte RNA. Waschen Sie es einmal mit 70% Ethanol, und an der Luft trocknen des Pellets. Das Pellet sollte rötlich. </li><li> Bereiten Sie eine Hybridisierungslösung mit 400 mM Na 2 HPO 4, pH 7,0, 0,8% BSA, 5% SDS, 12% Formamid 6 mit 1/15-1/100 ul des gereinigten, markierten Referenz-DNA (2,4) pro RNA-Probe. Die Menge der zugesetzten Referenz-DNA hängt von vielen verschiedenen Oligonukleotiden gekennzeichnet sind (2,4). Wenn es mehr als 100 sind, dann weniger Verdünnung erforderlich. </li><li> Lösen Sie das RNA-Pellet gut mit ~ 60 ul der Hybridisierungslösung. </li><li> Fügen Sie 20 ul Wasser zu jedem der Befeuchtung Brunnen in der Corning Microarray-Hybridisierung Kammer Basis. </li><li> Add ter Mischung aus markierter RNA und DNA-auf die Folie. Verwenden Sie einen dünnen Pipettenspitze, sanft berühren den Rand des lifterslip und lassen Sie die Lösung, um den Raum zwischen den lifterslip und schieben durch Saugen geben. </li><li> Legen Sie die Hybridisierung Kammer über die Basis abdecken und versiegeln Sie die Kammer mit Metallklammern. </li><li> Legen Sie die ganze Kammer in der Tüte, die mit der Kammer Kassette aus Corning kommt. </li><li> Legen Sie die Kassette Kammer (n) in einen Behälter mit benetzten Papiertuch; Abdeckung der Behälter mit Plastikfolie und legen Sie sie in einem 37 ° C feucht, Zellkulturbrutschrank für ~ 24 Stunden. Diese Vorsichtsmaßnahmen (3,6, 3,9, 3,10) sicherzustellen, dass die Hybridisierungslösung nicht austrocknet während der Inkubation. </li></ol> 4. Post-Hybridisierung Verarbeitung <ol><li> Demontieren der Kammer Kassetten eins nach dem anderen. Tauchen Sie ein Dia und seine lifterslip in 2 x SSC bei 22 ° C. Die lifterslip natürlich fallen die Folie. Legen Sie es in 0,8 x SSC. Waschendie Folien in 0,8 x SSC zweimal, dann dreimal in 0,4 x SSC, 1-2 min insgesamt. Trocknen Sie die Folien mit einer Zentrifuge mit einem Dia-Adapter. Waschen Sie die LifterSlips mit Wasser und sparen für die Wiederverwendung später. </li><li> Scannen Sie die Folien mit einem geeigneten Scanner, z. B. der Perkin Elmer ScanArray 5000 oder Molecular Devices Axon GenePix 4000B Mikroarray-Scanner. Scan bei Wellenlängen nahe 547 nm und 647 nm zu den entsprechenden Bild-Dateien zu erhalten. </li><li> Verwenden Sie Programme wie BlueFuse zu Pixelintensitäten in der Eingabe-, Bild-Dateien von 4,2) zu quantifizieren. Überprüfen Sie die einzelnen Spots mit dem Programm auf ungewöhnliche Flecken auf der Microarrays von der weiteren Betrachtung ausschließen. Diese abnormen Stellen entstehen in der Regel aus armen slide Druck oder Dia-Handling nach der Hybridisierung. </li><li> Verwenden Sie Programme wie GeneSpring und Excel für die Datenanalyse und-präsentation. Allgemeine Überlegungen für Microarray-Daten Normalisierung gelten, die über den Rahmen dieses Papiers. </li><li> Nach zufriedenstellenden Signale af erhaltenter 4,3), Streifen der Microarray-Slides zur Wiederverwendung 7. Spülen Sie die Folien in Wasser, dann tauchen in vorgewärmten, 1 mM NaOH und 0,1 x SSC in einer Färbung Schale bei 62 ° C für 10-20 Minuten. Wiederholen Sie die Inkubationszeit einmal. Waschen Sie die Slides ein paar Mal im Wasser für bis zu 60 Minuten unter leichtem Schütteln bei 22 ° C. Trocknen Sie die Folien mit einer Zentrifuge, und speichern Sie bei 22 ° C. </li><li> Zur Verwendung des regenerierten gleitet, ist es nicht notwendig, vor hybridisieren sie wieder. Einfach waschen Sie sie in Wasser durch Isopropanol folgte, und trocknen Sie die Dias rechts vor der Hybridisierung. </li></ol> 5. Repräsentative Ergebnisse: Wir haben vor allem die beschriebenen Verfahren gefolgt, um globale miRNA-Expression in Tausenden von Proben-Profil, dh RNAs vom Zebrafisch auf den menschlichen Proben isoliert unter vielen verschiedenen Bedingungen. Abbildung 1 zeigt das gescannte Bild eines Mikroarrays, sehr präzise und starke Hybridisierungssignale auf den Beweis gleiten. Die Pearson correlatiauf Koeffizienten zwischen technischen Replikate von Microarray-Hybridisierung sind ~ 0,99 7 zeigt eine hervorragende Reproduzierbarkeit. <img alt="Abbildung 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> Abbildung 1 Composite Bild einer gescannten miRNA Microarray-Rutsche nach der Hybridisierung. Rote Flecken resultierte aus Hybridisierungen von der Referenz-DNA, grünen Flecken aus dem DY547-markierte RNA-Probe, während die gelben Flecken wurden von Hybridisierungen sowohl von der DNA und RNA, die gleichen Sonden.

<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>

Keine Interessenskonflikte erklärt.

Trotz der jüngsten Fortschritte in der Deep-Sequencing-Technologien, bleibt Microarray eine gute Wahl für High-Throughput-Analyse von DNA und RNA. Im Vergleich zum tiefen Sequenzierung sind Microarray-Experimenten billiger, und ein typischer Molekularbiologie-Labor können die meisten der Experimente und Datenanalyse in-Haus, das für Flexibilität erlaubt und spart Zeit durchzuführen. In der Zukunft, Microarrays wahrscheinlich gut geeignet, um intensiv zu befragen Gruppen von Genen, z. B. sind, können alle oder ein...

<table border="1"><tbody><tr><th> Items </th><th> Verkäufer </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> NCode Multi-Species miRNA Microarray Probe Set V2 </td><td> Invitrogen </td><td> MIRMPS201 </td><td> Entwickelt auf Basis der miRBase Release 9.0 (Oktober 2006). Es enthält ~ 1.140 unverändert, 34-44 nt lange Oligonukleotide als Sonden für Wurm, Fliege, Zebrafisch, Maus, Ratte und Mensch miRNAs, und eine Reihe von internen Kontroll-Sonden wie snoRNAs. Die miRNA-Sonden werden Dubletten der Sequenzen komplementär zu reifen miRNAs, damit die Größe ~ 44 nt. Für die Analyse kann man auf miRNAs aus einer bestimmten Erbgut (s) von Interesse zu konzentrieren. </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596018 </td><td> Wir haben auch bereichert, kleine RNA-Fraktion für die Kennzeichnung, obwohl der Gesamt-RNA-Proben schneller und einfacher zu erstellen und zu quantifizieren und für nachgelagerte Anwendungen wie mRNA-Analyse sind. </td></tr><tr><td> T4 RNA Ligase 1 </td><td> New England Biolabs </td><td> M0204L </td><td></td></tr><tr><td> Ulysis Alexa Fluor 647 Nucleic Acid Labeling Kit </td><td> Invitrogen </td><td> U21660 </td><td> Dieses Kit oder ähnliche Produkte können verwendet werden, um experimentelle RNA-Proben oder ein Kontroll-RNA (anstelle von Kontroll-DNA) sowie Label werden. </td></tr><tr><td> 5&#39;-PCU-DY547-3 &#39; </td><td> Dharmacon </td><td> Custom made </td><td> Kleine RNA-Fraktion kann in ähnlicher Weise durch Ligation bezeichnet werden. </td></tr><tr><td> Centrisep Spalten </td><td> Princeton Separations </td><td> CS-901 </td><td></td></tr><tr><td> GAPSII beschichteten Objektträger </td><td> Corning </td><td> 40004 </td><td> Andere Arten von Folien können ebenfalls verwendet werden. </td></tr><tr><td> Microarray-Hybridisierung Kammern </td><td> Corning </td><td> 2551 oder 40080 </td><td> Andere Arten von Hybridisierung Kammern und Deckgläser sollten auch funktionieren. Mit kommerziell erhältlichen Hybridisierung Maschinen können Hybridisierung Zeit deutlich zu reduzieren, zB nach ~ 2 Stunden. </td></tr><tr><td> LifterSlips </td><td> ThErmo / 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

microRNAs (miRNAs) sind eine große Familie von ~ 22 Nukleotiden (nt) lange RNA-Moleküle, die weit in Eukaryoten 1 sind ausgedrückt. Komplexer Genome kodieren mindestens Hunderte von miRNAs, die in erster Linie hemmt die Expression einer Vielzahl von Zielgenen posttranskriptional 2, 3. miRNAs Steuerung einer breiten Palette von biologischen Prozessen 1. Darüber hinaus hat sich verändert miRNA-Expression mit menschlichen Krankheiten wie Krebs in Verbindung gebracht worden, und miRNAs können als Biomarker für Krankheiten und die Prognose 4, 5 dienen. Es ist daher wichtig, um die Expression und Funktion von miRNAs unter vielen verschiedenen Bedingungen zu verstehen. Real-time PCR, Microarray und deep sequencing: Drei große Ansätze zum Profil miRNA-Expression eingesetzt. Die Technik der miRNA-Microarray hat den Vorteil, dass High-Throughput, in der Regel weniger teuer, und die meisten der experimentellen und Analyseschritte können carr werdenin einem molekularbiologischen Labor an den meisten Universitäten, medizinischen Fakultäten und die damit verbundenen Krankenhäusern IED. Hier beschreiben wir ein Verfahren zur Durchführung benutzerdefinierte miRNA Microarray-Experimenten. Ein miRNA-Sonde gesetzt werden auf Glasträger gedruckt werden, um miRNA-Mikroarrays herzustellen. RNA isoliert mit einer Methode oder Reagenz, dass kleine RNA-Spezies konserviert und anschließend mit einem Fluoreszenz-Farbstoff markiert. Als Kontrolle werden Referenz-DNA-Oligonukleotide entsprechend einer Untergruppe von miRNAs auch mit einer anderen Fluoreszenz-Farbstoff markiert. Die Referenz-DNA wird dazu dienen, die Qualität der Folie und Hybridisierung zu demonstrieren und auch für Daten Normalisierung verwendet werden. Die RNA und DNA werden gemischt und hybridisierte mit einer Microarray-Folie mit Sonden für die meisten der miRNAs in der Datenbank. Nach dem Waschen wird die Folie gescannt, um Bilder zu erhalten, und Intensitäten der einzelnen Spots quantifiziert. Diese Roh-Signale werden weiter verarbeitet und analysiert werden, wie der Ausdruck von Daten der entsprechenden miRNAs. MicroArray Folien können entfernt und regeneriert werden, um die Kosten von Microarrays zu reduzieren und die Konsistenz der Microarray-Experimente zu verbessern. Die gleichen Grundsätze und Verfahren sind für andere Arten von benutzerdefinierten Microarray-Experimenten.

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

Performing Custom MicroRNA Microarray Experiments

Ein einfaches Verfahren zur Durchführung benutzerdefinierte microRNA Microarray-Experimenten beschrieben. Die Schritte umfassen Isolierung von RNA, die Kennzeichnung RNA und DNA-, Hybridisierung der Proben auf Microarrays, Scannen der Microarrays, Quantifizierung und Analyse Hybridisierungssignale.

Performing Benutzerdefinierte MicroRNA Microarray-Experimenten

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.5K Aufrufe.  University of Minnesota. Ein einfaches Verfahren zur Durchführung benutzerdefinierte microRNA Microarray-Experimenten beschrieben. Die Schritte umfassen Isolierung von RNA, die Kennzeichnung RNA und DNA-, Hybridisierung der Proben auf Microarrays, Scannen der Microarrays, Quantifizierung und Analyse Hybridisierungssignale.

Serie: 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 Ascosporen Produktion und Sammlung für Microarray-Experimente.

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

Forschung

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.

Herstellung künstlicher Doppelschichten für Elektrophysiologie Experimente

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.

Die Verarbeitung der 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 ...

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.

Globale Genexpressionsanalyse Mit einem Zebrafisch Oligonukleotid-Microarray-Plattform

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

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Measurement Of neuromagnetischen Hirnfunktionen Kinder im Vorschulalter mit benutzerdefinierten Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

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-Analyse für 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 mit CompoZr Benutzerdefinierte Zinkfinger Nukleasen (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 ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Reinigung und microRNA Profiling von Exosomen aus Blut und Kultur Medien Abgeleitet

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

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; Hybridisierung für Formalin fixiert Nierengewebe

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

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Verwendung Maßgeschneiderte Galvanotaxis Chambers zu Directional Migration Studie von Prostatazellen

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

Performing Benutzerdefinierte MicroRNA Microarray-Experimenten

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