Reverse-Transkriptase-Reaktion Schalten Heizblock auf 70-80 ° C und 42 ° C. Wasserbäder können auch in diesem Schritt verwendet werden. Wenn Sie Heizblöcke, setzte Wasser, so dass die Wärme gleichmäßig ist. <ol><li> Primingreaktion <ol><li> Take 3 pg RNA </li><li> Stellen Sie die Lautstärke auf 13 ul mit RNase freiem Wasser </li><li> Add 2,5 ul random Hexamer Primer (2mg/mL) </li></ol></li><li> Gut mischen und inkubieren bei 70-80 ° C für 8 Minuten. Dann, auf Eis für 5 min. </li><li> Bereiten RT-Cocktail (14,5 ul / rxn): <table border="0"><tbody><tr><td> Komponente </td><td> Volumen </td></tr><tr><td> 5X First Strand Buffer </td><td> 6 ul </td></tr><tr><td> 0,1 M DTT </td><td> 3 ul </td></tr><tr><td> 25X aminoallyl-dNTP-Mix </td><td> 1,2 ul </td></tr><tr><td> SuperScript II RT (200U/μl) </td><td> 2,3 ul </td></tr><tr><td> Wasser </td><td> 2,0 ul </td></tr></tbody></table> * Für n Reaktionen vorzubereiten Master Mix für n +0,5 Aliquots Cocktail, um sicherzustellen, dass Sie nicht ausgehen. </li><li> Add 14,5 ul gekühlt Reaktionen. </li><li> Mix (nicht vortexen) und inkubieren bei 42 ° C für 3-4 Stunden </li><li> Die Proben können bei -20 ° C gelagert werden, wenn nötig. </li></ol> Hydrolysieren RNA <ol><li> Zu jeder Probe, addiere 3 ul 1 N NaOH und 0,6 ul 0,5 M EDTA. (Final conc.s = 100mm NaOH, 10 mM EDTA) </li><li> Mix und Inkubation bei 65 ° C für 10 Minuten. </li><li> Neutralisieren, indem 33,6 ul 1M HEPES, pH = 7,0 (Endkonzentration 500 mM HEPES .=) </li><li> Die Proben können bei -20 ° C gelagert werden, falls erforderlich. </li></ol> Reaction Reinigung: Entfernung von Personengesellschaften aa-dUTP und freie Amine Hinweis: Diese Reinigung Protokoll wird von der Qiagen QIAquick PCRpurification Kit-Protokoll geändert. Die Phosphat-Wasch-und Elutionspuffer für die Qiagen mitgelieferten Puffern ersetzt, weil die Qiagen-Puffer freie Amine, die mit dem Cy-Farbstoff-Kupplungsreaktion konkurrieren enthalten. Die Spalten aus der Mini-Prep Kit kann auch verwendet werden. <ol><li> Mix cDNA-Reaktion mit 300 pl (oder 5x Reaktionsvolumen = 336 ul) Puffer PB (Qiagen mitgeliefert) und Transfer zum QIAquick Säule. </li><li> Legen Sie die Säule in ein 2 ml Sammel-Tube (Qiagen mitgeliefert) und Zentrifuge bei ~ 13.000 rpm für 1 Minute. Leere Sammelrohr. </li><li> Um Waschen 750 ul Phosphat Waschpuffer (nicht Qiagen) auf die Säule und zentrifugieren bei ~ 13.000 rpm für 1 Minute. </li><li> Leeren Sie den Deckel ab und wiederholen Sie die 750 ul waschen und Zentrifugationsschritt. </li><li> Leeren Sie den Deckel ab und Zentrifuge der Spalte 1 weitere Minute bei maximaler Geschwindigkeit. </li><li> Transfer-Säule in ein neues 1,5 mL Mikrozentrifugenröhrchen und vorsichtig 30 mu l Phosphat Elutionspuffer. (Siehe Lösung Vorbereitung Abschnitt) </li><li> Inkubation für 1 Minute bei Raumtemperatur. </li><li> Eluieren durch Zentrifugation bei ~ 13.000 rpm für 1 Minute. </li><li> Eluieren ein zweites Mal (mit einem anderen 30 ul Phosphat Elutionspuffer) in das gleiche Rohr durch Wiederholen der Schritte 6-8. Die endgültige Elutionsvolumen sollte ~ 60 ul werden. </li><li> Die Höhe der cDNA kann in diesem Stadium quantifiziert werden: ng cDNA = (Verdünnungsfaktor) (OD260) (37) (Gesamtvolumen von eluiert) </li><li> Dry Probe in einem Speed ​​Vac bei 45 ° C. </li><li> Die Proben können bei -20 ° C gelagert werden, wenn nötig. </li></ol> Coupling aa-cDNA zu Dye Ester Cy <ol><li> Resuspendieren aminoallyl-markierten cDNA mit 10 ul 50 mM Na-Bicarbonat, pH = 9,0, wenn nötig. (Dieser Puffer sollte frisches alle zwei Wochen durchgeführt werden) </li><li> Arbeiten an einem dunklen Ort, fügen Sie die Sonde an einem Schlauch der entsprechenden Cy-Farbstoff. Pipet nach oben und unten mehrmals zu resuspendieren des Farbstoffs. </li><li> Inkubieren Sie die Reaktion für 1 Stunde im Dunkeln bei Raumtemperatur. (Incubation kann bis zu 2 Stunden) </li></ol> Reaction Purification II: Entfernung von ungekoppelten Farbstoff <ol><li> Um die Reaktion hinzu 35 ul 100 mM NaOAc pH 5,2. Gründlich mischen. </li><li> Add 250 ul (oder 5x Reaktionsvolumen = 225 ul) Buffer PB (Qiagen im Lieferumfang enthalten) zu jeder Probe und mischen Sie durch Auf-und Abpipettieren. </li><li> Legen Sie eine QIAquick Spin-Säule in ein 2 ml Sammel-Tube (Qiagen mitgeliefert), die Probe auf die Säule und zentrifugieren bei ~ 13.000 für 1 Minute. Leere Sammelrohr. </li><li> Zum Waschen 0,75 ml Puffer PE (Qiagen mitgeliefert) an die Säule und zentrifugieren bei ~ 13.000 für 1 Minute. </li><li> Wiederholen Sie Schritt 4. </li><li> Leere Sammelröhrchen und Zentrifugationssäule für 1 weitere Minute bei maximaler Geschwindigkeit. </li><li> Legen Spalte in ein sauberes 1,5 mL Mikrozentrifugenröhrchen und vorsichtig 30 ul Puffer EB (Qiagen mitgeliefert) an die Mitte der Säule Membran. </li><li> Inkubation für 1 Minute bei Raumtemperatur. </li><li> Eluieren durch Zentrifugation bei ~ 13.000 rpm für 1 Minute. </li><li> Eluieren ein zweites Mal in das gleiche Rohr durch WiederholungSchritte 7-9. Der letzte eluierte Volumen sollte ~ 60 ul werden. </li></ol> Die Analyse der Labeling Reaction Die Höhe der cDNA und Label kann in diesem Stadium quantifiziert werden: <ul><li> Messen OD 260, 550 und 650 (Blank ist Wasser.) Verwenden Sie NanoDrop Microarray-Programm. </li><li> Berechnen Sie die pmol von Farbstoffen Einarbeitung und pmol DNA und nuc / Farbstoff-Verhältnis bei gegebener Gleichungen unter: pmol Nukleotiden = [OD260 * Volumen (ul) * 37 ng / ul * 1000 pg / ng] / 324,5 pg / pmol </li></ul> Anmerkung: 1 OD260 = 37 ng / ul für cDNA; 324,5 pg / pmol Molekulargewicht eines dNTP) pmol Cy3 = OD 550 * Volumen (ul) / 0,15 pmol Cy5 = OD650 * Volumen (ul) / 0,25 Nukleotide / Farbstoff-Verhältnis = pmol cDNA / pmol Cy Farbstoff Wenn Sie NanoDrop sind, es gibt bereits pmol / ul für jeden Farbstoff. Sie müssen nur diese Zahl nach Volumen zu multiplizieren, um insgesamt pmol Farbstoff Einarbeitung erhalten. Hinweis:&gt; 150-200 pmol des Farbstoffs Einbau pro Probe und einem Verhältnis von weniger als 20 Nukleotiden / Farbstoffmoleküle ist optimal für die Hybridisierungen. <ul><li> Die Proben können bei -20 ° C bei Dunkelheit gelagert werden. <ul><li> Kombinieren Sonden zusammen hybridisiert werden und trocken in Speedvac bei 42 ° C. </li><li> Die Proben können bei -20 ° C gelagert werden, bis bereit hyb. </li></ul></li></ul> Pre-hyb Dias <ol><li> Legen Sie schieben dem Gesicht nach unten über RT 100 ml Wasser in einem sauberen roten Diahalter für 3-5 Minuten </li><li> Snap trocken die Folie, indem Sie offen auf einem 100 ° C Heizblock für ein paar Sekunden - Uhr für die Kondensation zu verschwinden </li><li> UV Vernetzung der Folie bei 250 mJoules (Enter 2500 bis die UV-Vernetzer) </li><li> Hydrate gleitet durch Eintauchen in vorgewärmten 42 ° C Wasser. Spin 5 min bei 700 RPM bei RT. </li><li> Inkubieren in 50ml Coplin Gefäß mit vorgewärmten pre-hyb (5XSSC, 1% BSA, 0,1% SDS) für mindestens 45 Minuten bei 42 ° C, und die Inkubation kann länger gehen, wenn nötig. 50 ml pre-hyb Puffer: 12,5 ml 20x SSC, 10 ml 5% BSA, 0,5 ml 10% SDS, 27 ml Wasser </li><li> Dip Slides in vorgewärmte Wasser (42 ° C) und rühren ein paar Minuten vor hyb Puffer zu entfernen. </li><li> Spülen in Isopropanol und Spin 5 min bei 700 rpm, Raumtemperatur, um zu trocknen. </li></ol> Vorbereitung der Proben für die Hybridisierung <ol><li> Resuspendieren Sonde in Wasser (Bände sind unten angegeben) <table border="0"><tbody><tr><td> Für insgesamt vol. = 30μl </td><td> Für insgesamt vol. = 35μl </td><td> Für insgesamt vol .= 40 ul </td><td></td></tr><tr><td> 19,8 ul Wasser </td><td> 23,55 ul Wasser </td><td> 27,2 ul Wasser </td><td> </td></tr><tr><td> 1,5 ul </td><td> 1,5 ul </td><td> 1,5 ul </td><td> 10 mg / ml Lachssperma-DNA (Invitrogen) </td></tr><tr><td> 1,2 ul </td><td> 1,2 ul </td><td> 1,2 ul </td><td> 12,5 mg / ml tRNA </td></tr><tr><td> 4,5 ul </td><td> 5,25 ul </td><td> 6 ul </td><td> 20XSSC (3X endg.) </td></tr><tr><td> 3 ul </td><td> 3,5 ul </td><td> 4 ul </td><td> 1% SDS </td></tr></tbody></table> Hinweis: Die Reihenfolge der Zugabe der Reagenzien sind wichtig. Bereiten Sie nicht Mastermix. </li><li> 5 Minuten lang kochen, dann Spin 5 Minuten bei 14.000 Umdrehungen pro Minute. Cool für 2 min bei Raumtemp. </li></ol> Hybridisierung <ol><li> Add 10-12 ul von 3X SSC in die Vertiefungen der Hybridisierung Kammer an jeder Kante und in der Mitte. </li><li> Fügen Sie einen sauberen Heber Schlupf an den entsprechenden Bereich der Folie und sanft in Hybridisierungslösung Last (30 -40 ul). Es sollten zusätzliche Probe auf beiden Rand des Deckglases werden. </li><li> Legen Sie die Folie in der Kammer. Ziehen Sie die Schrauben und in einem 65 ° C Wasserbad über Nacht. Stellen Sie die Kammer direkt auf den Boden des Wasserbades. Legen Sie einen Block auf der Oberseite des obersten Kammer, alles in Position zu halten. Halten Sie den Deckel auf dem Wasserbad auf die lichtempfindliche Proben schützen. </li></ol> Hyb Wäscht <ol><li> Bereiten Sie die folgenden Puffer: <ul><li> 1X SSC, 0,05% SDS </li><li> 0,06 x SSC </li></ul></li><li> Nach der Inkubation entsiegeln Hybridisierung Kammer. Entfernen Sie die Folie aus der Kammer, dabei nicht zu dem Deckglas zu stören. </li><li> Um Deckgläser zu entfernen, tauchen Folien in einer Schale mit warmem 1X SSC, 0,05% SDS Waschpuffer Deckglas mit Blick auf den Boden der Schale. Das Deckglas fallen ab, indem Sie die Folie hin und her. </li><li> Nach dem Deckglas entfernt wird, legen Sie die Folie in einem Rack ein frisches Bad mit warmem 1XSSCm 0,05% SDS. Agitieren für ein paar Minuten </li><li> Objektträger und Rack, um ein Bad von 0,06 X SSC. </li><li> Objektträger zu einem anderen bath von 0,06 X SSC mit einem frischen Rack, Blotting die Folie auf ein Papiertuch, um so viel SDS-haltigem Puffer wie möglich, bevor Sie in den frischen Bad zu entfernen. </li><li> Agitieren Dias für ein paar Minuten. </li><li> Spin sofort bei Raumtemperatur für 5 min bei 700 rpm, gleitet dann sind bereit zum Scannen. Lagerung im Dunkeln, bis gescannt. </li></ol>

<ol>
	<li>Hasseman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Aminoallyl+Labeling+of+RNA+for.">TIGR Aminoallyl Labeling of RNA for.</a> Microarrays &amp; TIGR Microarray Labeled Probe Hybridization. , .</li><li>Gilbert, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Microbial+RNA+Aminoallyl+Labeling+for+Microarrays+&amp;+Hybridization+of+probed+labels.">TIGR Microbial RNA Aminoallyl Labeling for Microarrays &amp; Hybridization of probed labels.</a> , .</li><li>Hedge, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concise+guide+to+cDNA+Microarray+analysis-II.">A concise guide to cDNA Microarray analysis-II.</a> , .</li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AA-dUTP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0410</td>
<td>5-(3-aminoallyl)-2&#x2019;deoxyuridine-5&#x2019;-triphosphate</td>
</tr>
<tr>
<td>dNTP</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2035-01</td>
<td>100 mM dNTP Set PCR grade</td>
</tr>
<tr>
<td>Random Hexamer primers</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2166-01</td>
<td>3mg/mL</td>
</tr>
<tr>
<td>SuperScript III RT</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>80800444</td>
<td>200U/&#x03BC;L</td>
</tr>
<tr>
<td>CyDye&#x2122;</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN5661</td>
<td>Post-Labelling Reactive Dye Pack</td>
</tr>
<tr>
<td>QIAquick</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>PCR Purification kit</td>
</tr>
<tr>
<td>1M K2HPO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KH2PO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KPO4</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine:
9.5 mL 1M K2HPO4 and 0.5 ml 1M KH2PO4
</td>
</tr>
<tr>
<td>Phosphate wash buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 0.5 ml 1 M KPO4 pH 8.5 + 15.25 mL MilliQ water + 84.25 mL 95% ethanol. Wash buffer will be slightly cloudy.
** IMPORTANT: Phosphate wash buffer should be prepared daily.</td>
</tr>
<tr>
<td>Phosphate elution buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diluting 1 M KPO4, pH 8.5 to 4 mM with sterile water</td>
</tr>
<tr>
<td>Sodium Carbonate Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(Na2CO3): 0.5M, pH 9.0. Dissolve 4.2 g NaHCO3 in 80 mL of sterile water and adjust pH to 9.0 with 10 N NaOH; bring volume up to 100 mL with sterile water. To make a 50 mM solution for the dye coupling reaction dilute 1:10 with water.
Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month.</td>
</tr>		</tbody>
		</table>
		</div>

bacterial gene expression analysis using microarrays

Watch this Scientific Journal Video about Bacterial Gene Expression Analysis Using Microarrays at JoVE.com

Bacterial Gene Expression Analysis Using Microarrays

Bakterielle Gene Expression Analysis Using Microarrays

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

8.7K Aufrufe.  University of California Santa Cruz - UCSC. 

Serie: Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Forschung

Biologie

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis des Virus Gene Expression: Teil 3

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 ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Elektrophysiologie

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Analysis of Gene Expression in Emerald Ash Borer ( Agrilus planipennis) Mittels quantitativer Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Genomweite Analyse der Aminoacylierung (Charging) Ebenen der tRNA mittels Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Paraffin-Embedded-und Gefrierschnitte von Drosophila Adult Muskeln

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...