La reazione della trascrittasi inversa Girare blocco riscaldamento a 70-80 ° C e 42 ° C. Bagni di acqua può essere utilizzata anche in questo passaggio. Se si utilizza blocchi di riscaldamento, mettere un po &#39;d&#39;acqua in modo che il calore è uniforme. <ol><li> Adescamento di reazione <ol><li> Prendete 3 g di RNA </li><li> Regolare il volume a 13 microlitri con acqua RNasi libero </li><li> Aggiungere 2,5 ml di casuale esamero primer (2mg/ml) </li></ol></li><li> Mescolare bene e incubare a 70-80 ° C per 8 minuti. Poi, posto in ghiaccio per 5 min. </li><li> RT preparare cocktail (14,5 microlitri / RXN): <table border="0"><tbody><tr><td> Componente </td><td> Volume </td></tr><tr><td> 5X tampone prima parte </td><td> 6 microlitri </td></tr><tr><td> 0,1 M DTT </td><td> 3 ml </td></tr><tr><td> 25X aminoallyl-dNTP mix </td><td> 1,2 microlitri </td></tr><tr><td> SuperScript II RT (200U/μl) </td><td> 2,3 microlitri </td></tr><tr><td> Acqua </td><td> 2,0 microlitri </td></tr></tbody></table> * Fra le reazioni n, preparare Master Mix per n 0,5 aliquote di cocktail per assicurare che non si esaurirà. </li><li> Aggiungi 14,5 microlitri a reazioni raffreddato. </li><li> Mix (non vortex) e incubare a 42 ° C per 3-4 ore </li><li> I campioni possono essere conservati a -20 ° C se necessario. </li></ol> Idrolizzare l&#39;RNA <ol><li> Per ogni campione, aggiungere 3 ml di NaOH 1 N e 0,6 l di 0,5 M EDTA. (Final conc.s = 100mM NaOH, 10mM EDTA) </li><li> Miscelare e incubare a 65 ° C per 10 minuti. </li><li> Neutralizzare con l&#39;aggiunta di 33,6 microlitri HEPES 1M, pH = 7.0 (finale conc .= 500mm HEPES) </li><li> I campioni possono essere conservati a -20 ° C, se necessario. </li></ol> Reazione di purificazione: la rimozione di personalità giuridica aa-dUTP e ammine libero Nota: Questo protocollo di purificazione è modificata rispetto alla PCRpurification Qiagen QIAquick protocollo kit. Il lavaggio del fosfato e buffer di eluizione sono sostituiti i buffer Qiagen fornito perché i buffer Qiagen contengono ammine gratuito che competere con la reazione di accoppiamento Cy colorante. Le colonne dalla mini-prep kit può anche essere usato. <ol><li> Miscela di reazione cDNA con 300 ml (o il volume di reazione 5X = 336 mL) di buffer PB (Qiagen in dotazione) e trasferimento alla colonna QIAquick. </li><li> Porre la colonna in un tubo di raccolta da 2 ml (Qiagen in dotazione) e centrifugare a ~ 13.000 rpm per 1 minuto. Raccolta tubo vuoto. </li><li> Per lavare, aggiungere 750 microlitri di tampone di lavaggio fosfato (non è Qiagen) alla colonna e centrifugare a ~ 13.000 rpm per 1 minuto. </li><li> Svuotare il tubo di raccolta e ripetere il lavaggio 750 microlitri e fase di centrifugazione. </li><li> Svuotare il tubo di raccolta e centrifugare la colonna un ulteriore 1 minuto a velocità massima. </li><li> Trasferimento colonna in una nuova provetta da microcentrifuga 1,5 ml e con cautela, 30 microlitri di buffer di eluizione fosfato. (Vedi sezione preparazione soluzione) </li><li> Incubare per 1 minuto a temperatura ambiente. </li><li> Eluire per centrifugazione a ~ 13.000 rpm per 1 minuto. </li><li> Eluire una seconda volta (con un altro 30 microlitri di tampone fosfato eluizione) nel tubo stesso ripetendo i passaggi 6-8. Il volume di eluizione finale dovrebbe essere ~ 60 microlitri. </li><li> La quantità di cDNA può essere quantificata in questa fase: ng = cDNA (fattore di diluizione) (OD260) (37) (volume totale eluito dalla colonna) </li><li> Campione secco a una velocità di vac a 45 ° C. </li><li> I campioni possono essere conservati a -20 ° C se necessario. </li></ol> Accoppiamento aa-cDNA a Cy Ester Dye <ol><li> Risospendere aminoallyl cDNA marcato con 10 microlitri 50 mM Na-bicarbonato, pH = 9,0, se necessario. (Questo buffer dovrebbe essere preparata fresca ogni due settimane) </li><li> Lavorare in un luogo buio, aggiungere la sonda a un tubo del colorante Cy appropriato. Pipettare volte su e giù parecchie per sospendere di nuovo il colorante. </li><li> Incubare la reazione per 1 ora al buio a temperatura ambiente. (Incubazione può arrivare fino a due ore) </li></ol> Reazione Purificazione II: la rimozione di colorante disaccoppiato <ol><li> Per la reazione di aggiungere 35 microlitri NaOAc 100 mM pH 5.2. Mescolare accuratamente. </li><li> Aggiungere 250 microlitri (o il volume di reazione 5X = 225 mL) tampone PB (Qiagen in dotazione) per ogni campione e mescolare pipettando su e giù. </li><li> Inserire una colonna di spin QIAquick in un tubo di raccolta di 2 ml (Qiagen in dotazione), applicare il campione alla colonna e centrifugare a ~ 13.000 per 1 minuto. Raccolta tubo vuoto. </li><li> Per lavare, aggiungere 0,75 ml di tampone PE (Qiagen in dotazione) alla colonna e centrifugare a ~ 13.000 per 1 minuto. </li><li> Ripetere il punto 4. </li><li> Raccolta tubo vuoto e colonna centrifugare per un altro 1 minuto alla massima velocità. </li><li> Colonna Mettere in una provetta da microcentrifuga pulito 1,5 ml e con cautela, 30 microlitri tampone EB (Qiagen in dotazione) per il centro della membrana colonna. </li><li> Incubare per 1 minuto a temperatura ambiente. </li><li> Eluire per centrifugazione a ~ 13.000 rpm per 1 minuto. </li><li> Eluire una seconda volta nel tubo stesso ripetendoi passaggi 7-9. Il volume finale eluiti dovrebbe essere ~ 60 microlitri. </li></ol> Analisi di reazione di marcatura La quantità di cDNA e l&#39;etichetta può essere quantificata in questa fase: <ul><li> Misura OD 260, 550 e 650 (Blank è l&#39;acqua.) Utilizzare il programma NanoDrop microarray. </li><li> Calcolare la pmol di costituzione coloranti e DNA e nuc pmol / colorante rapporto con le equazioni sotto riportato: nucleotidi pmol = [OD260 volume * (mL) * 37 ng / mL * 1000 pg / ng] / 324,5 pg / pmol </li></ul> Nota: 1 OD260 = 37 ng / mL per cDNA; 324,5 pg / pmol peso molecolare medio di un dNTP) pmol di Cy3 OD550 * = volume (mL) / 0.15 pmol Cy5 OD650 * = volume (mL) / 0.25 nucleotidi / rapporto colorante cDNA = pmol / pmol Cy colorante Se si utilizza NanoDrop, dà già pmol / mL per ogni tinta. Hai solo bisogno di moltiplicare questo numero per volume di ottenere totale incorporazione pmol colorante. Nota:&gt; 150-200 pmol di costituzione colorante per campione e un rapporto di meno di 20 nucleotidi / molecole di colorante è ottimale per ibridazioni. <ul><li> Campioni possono essere conservati a -20 ° C al buio. <ul><li> Combinano sonde di essere ibridato insieme e asciutto nel speedvac a 42 ° C. </li><li> I campioni possono essere conservati a -20 ° C fino al momento di Ibr. </li></ul></li></ul> Pre-Hyb diapositive <ol><li> Faccia scivolare verso il basso posto oltre RT 100 ml di acqua in un supporto pulito scivolo rosso per 3-5 minuti </li><li> Scatto secco la diapositiva mettendo a faccia in su di 100 ° C il riscaldamento blocco per alcuni secondi - guarda per la condensazione a sparire </li><li> UV cross-link la diapositiva a 250 mJoules (Inserisci il 2500 a reticolante UV) </li><li> Diapositive idrato per immersione in pre-riscaldato acqua 42 ° C. Spin 5 min a 700 giri a temperatura ambiente. </li><li> Diapositive incubare in vasetto da 50 ml contenente Coplin preriscaldata pre-Ibr (5XSSC, BSA 1%, 0,1% SDS) per almeno 45 minuti a 42 ° C, e l&#39;incubazione può andare più a lungo se necessario. 50 ml di pre-Hyb tampone: 12,5 mL 20x SSC, 10 ml 5% di BSA, 0,5 ml 10% SDS, 27 ml di acqua </li><li> Immergere i vetrini in pre-riscaldato acqua (42 ° C) e agitare un paio di minuti per eliminare pre-Hyb buffer. </li><li> Sciacquare in isopropanolo e spin 5 min a 700 rpm, temperatura ambiente, ad asciugare. </li></ol> Preparazione dei campioni per l&#39;ibridazione <ol><li> Risospendere sonda in acqua (volume sono riportati di seguito) <table border="0"><tbody><tr><td> Per un totale vol. = 30μl </td><td> Per un totale vol. = 35μl </td><td> Vol .= per un totale di 40μl </td><td></td></tr><tr><td> 19,8 microlitri di acqua </td><td> 23,55 di acqua microlitri </td><td> 27,2 microlitri di acqua </td><td> </td></tr><tr><td> 1,5 microlitri </td><td> 1,5 microlitri </td><td> 1,5 microlitri </td><td> 10 mg / ml di DNA dello sperma di salmone (Invitrogen) </td></tr><tr><td> 1,2 microlitri </td><td> 1,2 microlitri </td><td> 1,2 microlitri </td><td> 12,5 mg / ml tRNA </td></tr><tr><td> 4,5 microlitri </td><td> 5,25 microlitri </td><td> 6 microlitri </td><td> 20XSSC (3X finale) </td></tr><tr><td> 3 ml </td><td> 3,5 microlitri </td><td> 4 microlitri </td><td> 1% SDS </td></tr></tbody></table> Nota: L&#39;ordine di aggiunta dei reagenti sono importanti. Non preparare master mix. </li><li> Far bollire per 5 minuti, poi girare 5 minuti a 14.000 giri al minuto. Raffreddare per 2 minuti a temperatura ambiente. </li></ol> Ibridazione <ol><li> Aggiungere 10-12 ml di SSC 3X per i pozzi della camera di ibridazione su ogni bordo e al centro. </li><li> Aggiungi una scivolata pulita sollevatore per l&#39;area appropriata della diapositiva e delicatamente carico in soluzione di ibridazione (30 -40 microlitri). Ci dovrebbe essere campione aggiuntivo su entrambi i bordi del vetrino. </li><li> Mettere il vetrino nella camera. Serrare le viti e mettere in un bagno di acqua a 65 ° C durante la notte. Non posizionare la camera direttamente sul fondo della vasca d&#39;acqua. Posizionare un blocco sulla parte superiore della camera superiore a tenere tutto a posto. Tenere il coperchio sul bagno in acqua per proteggere i campioni sensibile alla luce. </li></ol> Ibr Lava <ol><li> Preparare i buffer seguenti: <ul><li> SSC 1X, 0,05% SDS </li><li> 0,06 X SSC </li></ul></li><li> Dopo la camera di ibridazione dissigillare incubazione. Rimuovere il vetrino dalla camera, facendo attenzione a non disturbare il coprioggetto. </li><li> Per rimuovere coprioggetto, scivoli immergere in un piatto caldo contenente SSC 1X, 0,05% SDS coprioggetto tampone di lavaggio di fronte al fondo del piatto. Il coprioggetto cadranno spostando il lato scorrimento a lato. </li><li> Dopo che il coprioggetto viene rimosso, posizionare il vetrino in un rack con un bagno fresco caldo 1XSSCm 0,05% SDS. Agitare per un paio di minuti </li><li> Trasferimento diapositive più rack per un bagno di 0,06 X SSC. </li><li> Trasferimento diapositive ad un altro ba° di 0,06 X SSC contenente un rack fresca, cancellando la diapositiva su un tovagliolo di carta per rimuovere il più SDS-contenente tampone il più possibile prima di mettere nella vasca da bagno fresco. </li><li> Agitare scivoli per un paio di minuti. </li><li> Girare subito a temperatura ambiente per 5 min a 700 rpm, poi scivola pronti per la scansione. Conservare al buio fino a scansione. </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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		<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

Batterica analisi di espressione genica mediante microarray

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

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

Video: 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.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

Ricerca

Biologia

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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 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.

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 ...

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 Elettrofisiologia

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

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.

Analisi dell&#39;espressione genica in Borer Emerald Ash ( Agrilus planipennis) Utilizzando quantitativa 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.

Genoma Analisi dei Aminoacylation (carica) I livelli di tRNA usando i 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.

Sezioni incluse in paraffina e surgelati di Drosophila Adulti Muscoli

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

Methods Article

Batterica analisi di espressione genica mediante microarray