University of Vermont, Vermont Genetics Network. This publication was made possible by the Vermont Genetics Network, through Grant Number P20 RR16462 from the BRIN Program and Grant Number P20 RR16462 from the INBRE Program of theNational Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.
We would like to thank all our participating outreach institutions for their collaboration and input in refining this module Castleton State College, Green Mountain College, Johnson State College, Lyndon State College, Marlboro College, Middlebury College, Norwich University and Saint Michael's College.

<ol>
	<li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc Natl Acad Sci U S A. 87, 1663-1667 (1990).</li><li>Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+expression+programs+in+the+response+of+yeast+cells+to+environmental+changes.">Genomic expression programs in the response of yeast cells to environmental changes.</a> Mol Biol Cell. 11, 4241-4257 (2000).</li><li>D'Autr&#233;aux, B., Toledano, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+as+signalling+molecules:+mechanisms+that+generate+specificity+in+ROS+homeostasis.">ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.</a> Nat Rev Mol Cell Biol. 8, 813-824 (2007).</li><li>Shenton, D., Smirnova, J. B., Selley, J. N., Carroll, K., Hubbard, S. J., Pavitt, G. D., Ashe, M. P., Grant, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+translational+responses+to+oxidative+stress+impact+upon+multiple+levels+of+protein+synthesis.">Global translational responses to oxidative stress impact upon multiple levels of protein synthesis.</a> J Biol Chem. 281, 29011-29021 (2006).</li><li>Godon, C., Lagniel, G., Lee, J., Buhler, J. M., Kieffer, S., Perrot, M., Boucherie, H., Toledano, M. B., Labarre, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+H2O2+stimulon+in+Saccharomyces+cerevisiae.">The H2O2 stimulon in Saccharomyces cerevisiae.</a> J Biol Chem. 273, 22480-229 (1998).</li><li>Vermont Genetics Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microarray Outreach. , (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Affymetrix GeneChip Expression Analysis: Technical Manual. , (2004).</li><li>Huang, D. W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+Bioinformatics+Resources.">Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources.</a> Nature Protoc. 4, 44-57 (2009).</li><li>Dennis, G., Sherman, B. T., Hosack, D. A., Yang, J., Gao, W., Lane, H. C., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAVID:+Database+for+Annotation,+Visualization,+and+Integrated+Discovery.">DAVID: Database for Annotation, Visualization, and Integrated Discovery.</a> Genome Biol. 4, P3-P3 (2003).</li></ol>

The production of this video-article was sponsored by EMD-Millipore.

Applications and significance.
The Vermont Genetics Network outreach program, at the University of Vermont, conducts undergraduate outreach to eight partner baccalaureate colleges throughout the state. The goal of the VGN Outreach Core is to expose undergraduates in the state of Vermont to state-of-the-art scientific technology &amp; resources using hands-on experiences. The microarray module described was developed in 2003 and subsequently upgraded. It has been offered as a mini-course or integrated into existing laboratory curriculum at the participating institutions.
This project, between research university cores and undergraduate colleges, promotes collaborations in education and research. Microarray outreach throughout the state has enhanced curriculum and created research and networking opportunities for core facilities, faculty and students.
As undergraduate faculty adopt the program they are encouraged to design unique experiments provided there are appropriate Affymetrix GeneChips and annotation available. All information for this module including the laboratory manual, references and PowerPoint presentations are available online (Vermont Genetics Network, 2008).
Critical steps:
Spheroplasting: It is important to observe successful spheroplasting under the microscope. The use of SDS is highly beneficial as it causes the yeast swelling resulting in spheroids. It is important to observe that the budding cells form spheroids as well. If partial spheroplasting is observed, an extended treatment with lyticase is recommended.
Pellet cleaning: During the precipitation reaction and subsequent ethanol washing step, it is very easy to lose the cDNA pellet. Extreme care and meticulous attention to the pellet is imperative. 
Applying the water to the center of the membrane: During the RNA elution step from the membrane, it is important to "squirt" the 30 &mu;l of water directly to the center of the silica membrane. If this is not accomplished, the column can be centrifuged and the resulting elutant can be re-applied to the silica membrane again. This can be repeated as many times as necessary.
Modifications and Product Substitutions:
<ol>
 <li>Alternative RNA clean-up columns can be substituted. Examples are the USB prep-ease and the Invitrogen Pure Link RNA kit, and the Omega RNA clean-up kit to name a few.</li>
 <li>Schizosaccharomyces pombe is an optional yeast. The Affymetrix GeneChip contains both genomes on the same chip</li>
 <li>Various treatments and times may be used. This may include drug treatments, temperature treatments, anti-oxidants, etc. Treatment times may also be adjusted.</li>
 <li>DNase treatment may not be required because the methods described employ the use of an Oligo (dT) primer.</li>
 <li>It is not required to perform a double elution of the RNA off the column with the same 30 &mu;l aliquot. Once is almost always adequate. This is suggested to insure that a complete recovery is obtained. Additionally, the water used to elute the RNA from the column can be pre-heated to 65&deg;C to further enhance the ability to recover RNA from the silica column.</li>
 <li>Many methods are available to quantify RNA. The Eppendorf and Nanodrop have only been selected for their ease of use and tested accuracy.</li>
 <li>Agarose gel electrophoresis many be performed using standard laboratory equipment. The E-Gel is not required but is desirable because it is RNase free and does not require gel solidification waiting time.</li>
 <li>Ordering information for products has been supplied. However, many general reagents can be ordered from multiple vendors.</li>
 <li>One Cycle cDNA reagents can be purchased separately if that is more cost effective.</li>
 <li>There are other target prep methods but we feel this one offers more teaching opportunities for the student.</li>
</ol>

<table border="1">
	<tbody>
		<tr>
			<td>General Laboratory Equipment</td><td>Company</td><td>Product number</td></tr>
		<tr>
			<td>Spetrophotometer</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Thermocyler</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Microcentrifuge</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Vortex</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Stir Plates (2)</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-10s pipettors</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-20's pipettors:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-200's pipettors:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>P-1000's pipettors:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Camera and transilluminator</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td colspan="3">&nbsp;</td></tr>
		<tr>
			<td>Supplies - recommended</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E-Gel Apparatus</td><td>Invitrogen</td><td>G6000-08</td></tr>
		<tr>
			<td>E-Gels</td><td>Invitrogen</td><td>G6018-02</td></tr>
		<tr>
			<td>Axygen 1.7 ml. Clear</td><td>Krackler</td><td>383-MCT175C</td></tr>
		<tr>
			<td>Axygen 0.5 ml clear tubes</td><td>Krackler</td><td>383-MCT060C</td></tr>
		<tr>
			<td>Axygen 2ml capture tubes</td><td>Krackler</td><td>383-MCT200NC</td></tr>
		<tr>
			<td>boxes of P-10 ART tips:</td><td>CLP</td><td>BT10XL</td></tr>
		<tr>
			<td>Boxes of P-100 ART tips:</td><td>CLP</td><td>BT200</td></tr>
		<tr>
			<td>Boxes of P-20 ART Tips:</td><td>CLP</td><td>BT20</td></tr>
		<tr>
			<td>Boxes of P-1000 ART Tips:</td><td>CLP</td><td>BT1000</td></tr>
		<tr>
			<td>Yeast Chips</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td colspan="3">&nbsp;</td></tr>
		<tr>
			<td>General Laboratory Supplies</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>25 ml sterile disposable pipets-</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>5ml sterile disposable pipets-</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Microfuge tube racks</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>KimWipes:</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Lab Coats</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Stir Bars</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Culture flasks</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Innoculating loops</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Sharpies</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Gloves</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Autoclave tape</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Stirrer bars</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td colspan="3">&nbsp;</td></tr>
		<tr>
			<td>Reagents</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>30% Hydrogen Peroxide</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-386790?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>386790</td></tr>
		<tr>
			<td>HBSS without Ca, Mg, or dye</td><td>Sigma-Aldrich</td><td>H6648-100ml</td></tr>
		<tr>
			<td>Qiagen Rneasy Mini Kit:</td><td>Qiagen</td><td>74104</td></tr>
		<tr>
			<td>Qiagen DNase Kit:</td><td>Qiagen</td><td>79254</td></tr>
		<tr>
			<td>Rnase Remover</td><td>Denville Scientific</td><td>D1180</td></tr>
		<tr>
			<td>Harleco Alcohol 100%</td><td><a href="http://www.emdchemicals.com/harleco-alcohol/EMD_CHEM-65347/p_uuid?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>65347</td></tr>
		<tr>
			<td>ENZO IVT Kit</td><td>ENZO</td><td>ENZ-42655-10</td></tr>
		<tr>
			<td>Lyticase: one bottle</td><td>VWR</td><td>IC19012310</td></tr>
		<tr>
			<td>Water, Cell Culture grade</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-4.86505?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>4.86505</td></tr>
		<tr>
			<td>Yeast culture:</td><td>ATCC</td><td>18824</td></tr>
		<tr>
			<td>YPD broth powder</td><td><a href="http://www.emdchemicals.com/products/EMD_CHEM-4.85040?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>4.8504</td></tr>
		<tr>
			<td>D(+) Glucose, Anhydrous</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-346351?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>346351</td></tr>
		<tr>
			<td>Sorbitol</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-56755?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>56755</td></tr>
		<tr>
			<td>EDTA</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-4005?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>4005</td></tr>
		<tr>
			<td>SDS solution, 20%</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-7990-OP?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>7990-OP</td></tr>
		<tr>
			<td>Pellet Paint</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-69049?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>69049</td></tr>
		<tr>
			<td>PCI RNase DNase free (7 aliquots):</td><td>Fisher</td><td>AC32711-500</td></tr>
		<tr>
			<td>7.5M NH4OAc</td><td>Fisher</td><td>ICN1987 5980</td></tr>
		<tr>
			<td>Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA)</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Trizma Base</td><td>Fiaher</td><td>50-899-90034</td></tr>
		<tr>
			<td>KOAc</td><td>Fisher</td><td>NC9757725</td></tr>
		<tr>
			<td>MgOA</td><td>Fisher</td><td>NC9681042</td></tr>
		<tr>
			<td>Loading Dye</td><td>Invitrogen</td><td>10482055</td></tr>
		<tr>
			<td>PCR Markers</td><td><a href="http://www.merck-chemicals.com/products/EMD_BIO-69278-3?WT.mc_id=2usen000105
">EMD Millipore</a></td><td>69278-3</td></tr>
		<tr>
			<td>Phase Lock Gels</td><td>Fisher</td><td>FP2302820</td></tr>
		<tr>
			<td>One-Cycle cDNA Kit</td><td>Invitrogen</td><td>A10752-030</td></tr>
		<tr>
			<td>Includes;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E. coli DNA Pol</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>dNTP's</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>T4 DNA Pol.</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>T-7 oligod(T)</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>0.5ml EDTA pH 8.0</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>First Strand Buffer</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E. Coli RNaseH</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>Second Strand Buffer</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>E. Coli DNA ligase</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>SuperScript II</td><td>&nbsp;</td><td>&nbsp;</td></tr>
		<tr>
			<td>DTT</td><td>&nbsp;</td><td>&nbsp;</td></tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Microarray Analysis for Saccharomyces cerevisiae at JoVE.com

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 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 ...

microarray-analysis-for-saccharomyces-cerevisiae

Research

JoVE Journal

Biology

13.4K vues.  The University of Vermont. Dans ce protocole d'expression génique, chez la levure ( Saccharomyces cerevisiae) Est modifié après l'exposition au stress oxydatif induit par l'ajout de peroxyde d'hydrogène (H 2 O 2), Un agent oxydant.