Name: Microarray Analysis for Saccharomyces cerevisiae (Video) | JoVE Journal
Uploaded: 2011-04-07T13:00:00.000+00:00
Duration: 13 min 17 s
Description: Learn microarray analysis using RNA extraction, cDNA synthesis, and bioinformatics in Saccharomyces cerevisiae. This computational method teaches gene expression profiling under oxidative stress to undergraduate students.

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5 minutes

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/pubmed?term=((Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Genomic+expression+programs+in+the+response+of+yeast+cells+to+environmental+changes%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Genomic expression programs in the response of yeast cells to environmental changes.</a> Mol Biol Cell. 11, 4241-4257 (2000).</li>
<li>D'Autréaux, B., Toledano, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ROS+as+signalling+molecules%3A+mechanisms+that+generate+specificity+in+ROS+homeostasis%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Global+translational+responses+to+oxidative+stress+impact+upon+multiple+levels+of+protein+synthesis%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((The+H2O2+stimulon+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">The H2O2 stimulon in Saccharomyces cerevisiae.</a> J Biol Chem. 273, 22480-229 (1998).</li>
<li>Vermont Genetics Network. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Microarray+Outreach%22">Microarray Outreach</a>. , (2008).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Affymetrix+GeneChip+Expression+Analysis%3A+Technical+Manual%22">Affymetrix GeneChip Expression Analysis: Technical Manual</a>. , Affymetrix. Santa Clara, CA. (2004).</li>
<li>Huang, D. W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+Bioinformatics+Resources%5BTitle%5D)+AND+%22Nature+Protoc%22%5BJournal%5D)">Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources.</a> Nature Protoc. 4, 44-57 (2009).</li>
<li>Dennis, G. Jr, 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/pubmed?term=((DAVID%3A+Database+for+Annotation%2C+Visualization%2C+and+Integrated+Discovery%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">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><tbody><tr><td>Spetrophotometer</td><td></td><td></td><td></td></tr><tr><td>Thermocyler</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>Stir Plates (2)</td><td></td><td></td><td></td></tr><tr><td>P-10s pipettors</td><td></td><td></td><td></td></tr><tr><td>P-20&amp;#39;s pipettors:</td><td></td><td></td><td></td></tr><tr><td>P-200&amp;#39;s pipettors:</td><td></td><td></td><td></td></tr><tr><td>P-1000&amp;#39;s pipettors:</td><td></td><td></td><td></td></tr><tr><td>Camera and transilluminator</td><td></td><td></td><td></td></tr><tr><td>E-Gel Apparatus</td><td>Invitrogen</td><td>G6000-08</td><td></td></tr><tr><td>E-Gels</td><td>Invitrogen</td><td>G6018-02</td><td></td></tr><tr><td>Axygen 1.7 ml. Clear</td><td>Krackeler Scientific, Inc.</td><td>383-MCT175C</td><td></td></tr><tr><td>Axygen 0.5 ml clear tubes</td><td>Krackeler Scientific, Inc.</td><td>383-MCT060C</td><td></td></tr><tr><td>Axygen 2ml capture tubes</td><td>Krackeler Scientific, Inc.</td><td>383-MCT200NC</td><td></td></tr><tr><td>boxes of P-10 ART tips:</td><td>CLP</td><td>BT10XL</td><td></td></tr><tr><td>Boxes of P-100 ART tips:</td><td>CLP</td><td>BT200</td><td></td></tr><tr><td>Boxes of P-20 ART Tips:</td><td>CLP</td><td>BT20</td><td></td></tr><tr><td>Boxes of P-1000 ART Tips:</td><td>CLP</td><td>BT1000</td><td></td></tr><tr><td>Yeast Chips</td><td></td><td></td><td></td></tr><tr><td>25 ml sterile disposable pipets-</td><td></td><td></td><td></td></tr><tr><td>5ml sterile disposable pipets-</td><td></td><td></td><td></td></tr><tr><td>Microfuge tube racks</td><td></td><td></td><td></td></tr><tr><td>KimWipes:</td><td></td><td></td><td></td></tr><tr><td>Lab Coats</td><td></td><td></td><td></td></tr><tr><td>Stir Bars</td><td></td><td></td><td></td></tr><tr><td>Culture flasks</td><td></td><td></td><td></td></tr><tr><td>Innoculating loops</td><td></td><td></td><td></td></tr><tr><td>Sharpies</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Autoclave tape</td><td></td><td></td><td></td></tr><tr><td>Stirrer bars</td><td></td><td></td><td></td></tr><tr><td>30% Hydrogen Peroxide</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>386790</td><td></td></tr><tr><td>HBSS without Ca, Mg, or dye</td><td>Sigma-Aldrich</td><td>H6648-100ml</td><td></td></tr><tr><td>Qiagen Rneasy Mini Kit:</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Qiagen DNase Kit:</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Rnase Remover</td><td>Denville Scientific</td><td>D1180</td><td></td></tr><tr><td>Harleco Alcohol 100%</td><td><a href="http://www.emdchemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>65347</td><td></td></tr><tr><td>ENZO IVT Kit</td><td>Enzo Life Sciences</td><td>ENZ-42655-10</td><td></td></tr><tr><td>Lyticase: one bottle</td><td>VWR international</td><td>IC19012310</td><td></td></tr><tr><td>Water, Cell Culture grade</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>4.86505</td><td></td></tr><tr><td>Yeast culture:</td><td>ATCC</td><td>18824</td><td></td></tr><tr><td>YPD broth powder</td><td><a href="http://www.emdchemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>4.8504</td><td></td></tr><tr><td>D(+) Glucose, Anhydrous</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>346351</td><td></td></tr><tr><td>Sorbitol</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>56755</td><td></td></tr><tr><td>EDTA</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>4005</td><td></td></tr><tr><td>SDS solution, 20%</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>7990-OP</td><td></td></tr><tr><td>Pellet Paint</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>69049</td><td></td></tr><tr><td>PCI RNase DNase free (7 aliquots):</td><td>Fisher Scientific</td><td>AC32711-500</td><td></td></tr><tr><td>7.5M NH4OAc</td><td>Fisher Scientific</td><td>ICN1987 5980</td><td></td></tr><tr><td>Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA)</td><td></td><td></td><td></td></tr><tr><td>Trizma Base</td><td>Fiaher</td><td>50-899-90034</td><td></td></tr><tr><td>KOAc</td><td>Fisher Scientific</td><td>NC9757725</td><td></td></tr><tr><td>MgOA</td><td>Fisher Scientific</td><td>NC9681042</td><td></td></tr><tr><td>Loading Dye</td><td>Invitrogen</td><td>10482055</td><td></td></tr><tr><td>PCR Markers</td><td><a href="http://www.merck-chemicals.com" target="_blank"><img src="/img/new_window.png"> EMD Millipore</a></td><td>69278-3</td><td></td></tr><tr><td>Phase Lock Gels</td><td>Fisher Scientific</td><td>FP2302820</td><td></td></tr><tr><td>One-Cycle cDNA Kit</td><td>Invitrogen</td><td>A10752-030</td><td></td></tr><tr><td>Includes;</td><td></td><td></td><td></td></tr><tr><td>E. coli DNA Pol</td><td></td><td></td><td></td></tr><tr><td>dNTP&amp;#39;s</td><td></td><td></td><td></td></tr><tr><td>T4 DNA Pol.</td><td></td><td></td><td></td></tr><tr><td>T-7 oligod(T)</td><td></td><td></td><td></td></tr><tr><td>0.5ml EDTA pH 8.0</td><td></td><td></td><td></td></tr><tr><td>First Strand Buffer</td><td></td><td></td><td></td></tr><tr><td>E. Coli RNaseH</td><td></td><td></td><td></td></tr><tr><td>Second Strand Buffer</td><td></td><td></td><td></td></tr><tr><td>E. Coli DNA ligase</td><td></td><td></td><td></td></tr><tr><td>SuperScript II</td><td></td><td></td><td></td></tr><tr><td>DTT</td><td></td><td></td><td></td></tr></tbody></table>

microarray analysis for saccharomyces cerevisiae

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.

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 this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

The overall goal of this procedure is to introduce undergraduate science students to microarray technology by examining the expression differences in yeast undergoing oxidative stress. This is accomplished by first isolating total RNA from yeast cultures treated with hydrogen peroxide and the untreated controls. The second step of the procedure is to generate and purify CDNA from these RNA samples.The CDNA is then used to prepare biotin labeled CRNA via in vitro transcription. The final step of the procedure is fragmentation of the biotin labeled CRNAs for hybridization to the AFI metrics yeast gene chips. Ultimately, results can be obtained that show the expression differences in the two yeast samples and through bioinformatics demonstrate the power of microarray analysis to undergraduate students.The Vermont Genetics Network Outreach team first had the idea for this method when they were charged with delivering microray technology to science students throughout the state of Vermont in order to enhance their science education. To date this method has been delivered to multiple sites at eight baccalaureate colleges throughout the state. So the main advantage of using microray of at teaching modules, it gives us the ability to teach general molecular biology techniques that are used every day throughout the lab to a group of individuals.And we also get to use this to teach to students and teachers alike and our groups. We actually have students and professors working together on the same project. They learn certain techniques that take a lot of finesse, like handling RNA, which is not an easy task.They learn how to precipitate DNA using visualization reagents. They actually get to use the techniques that they're gonna encounter in graduate studies or in everyday molecular biology labs. Though this method is suitable for gaining insight into oxidative stress and yeast, it's certainly applicable to other model organisms and other biological systems that you may wanna look at.Gene expression changes whether they're associated with developmental changes, environmental toxins, specific disease states, and really many more. To examine the expression differences in yeast undergoing oxidative stress, total RNA is first extracted from two yeast cultures by enzymatic lysis. One culture was exposed to oxidative stress by a one hour treatment with 0.5 millimolar hydrogen peroxide in hank's buffered saline while the other culture was treated with only HBSS as a control.Begin by labeling two 1.7 milliliter micro centrifuge tubes with the appropriate identification information. Transfer 1.5 milliliters of the appropriate yeast culture into each tube. Centrifuge the tubes at 5, 000 Gs for two minutes at room temperature.After centrifugation, use a micro pipette to carefully remove and discard the supernatant without disturbing the pellet. Check that the pellet is big enough before continuing with the procedure. If the pellet is too small, add 1.5 milliliters of culture into the same tube containing the pellet and then centrifuge to obtain a bigger pellet.Discard the supernatant with a micro pipette, removing as much of the liquid as possible from the yeast pellet. Then add 100 microliters of SG buffer and 30 microliters of liase solution to the yeast pellet vortex to mix. Incubate both tubes for 30 minutes at room temperature during the incubation.Gently swirl each tube every 10 minutes to generate plats. One of the most critical steps when you're doing sphero plating of yeast is to make sure that you actually created a hundred percent Sphero plast after the treatment. That means not all lytic enzyme is created equal.So you have to check to make sure good lytic enzyme is giving you good sphero plating, meaning you have to take these samples, put them onto a microscope slide and check under the microscope by adding 0.1%SDS to ensure that you do have protoplasts formation. To observe complete sphero plating, pipette 10 microliters of the yeast sample onto a microscope slide while examining the sample under the microscope at one microliter of 0.1%SDS to cause the yeast cells to swell and form perfect spheres or spheroids. It is important to observe that the budding cells form S spheroids as well.If partial sphero plating is observed, an extended treatment with liase is recommended. Once complete sphero plating has been confirmed at 350 microliters of BRLT buffer to each tube, then add 250 microliters of 100%ethanol. Ensure the tube is tightly capped by holding the lid closed and vortex vigorously for one minute.This procedure will lice the Sphero plats. After this, use R and easy Z spin columns to collect and clean the RNA from the two cultures. Finally, evaluate the quality of the extracted RNA using 1.2%precast aros gel for electrophoresis to prepare biotin labeled CRNA.The total RNA extracted from the yeast cells is first used to synthesize CD NA by reverse transcription. After CD NA generation prepare phase lock gel tubes for use in CD NA precipitation centrifuge phase lock gel tubes at full speed for one minute to ensure the gel is at the bottom of the tube. Do not vortex phase lock tubes to precipitate CD NA pipette 162 microliters of the bottom layer of a pH 8.0 tris buffered phenyl chloroform, isoamyl alcohol, or PCI mixture and add to the 162 microliter contents of the second strand, CD NA synthesis reaction tube equal volumes of aqueous and organic mixtures are required for this step.Vortex for five seconds to mix the contents. Next, use a micro pipette transfer all of the CD N-A-P-C-I mixture to the phase lock gel tube. Do not vortex the phase lock gel tube centrifuge at full speed for two minutes.Then use a micro pipette to transfer the top layer from the phase lock gel tube to a newly labeled 1.7 milliliter tube. Try to collect as much of the layer as possible. Add the following to the 1.7 milliliter micro centrifuge tube, 405 microliters of 100%ethanol, 80 microliters of ammonium acetate, and one microliter of pellet paint vortex.Briefly place the tube in the centrifuge with the hinge of the tube facing out and centrifuge at full speed for 20 minutes. At room temperature when centrifugation is complete, gently remove the tube from the centrifuge being careful not to disturb the CD NA pellet. The pellet should be pink because of the pellet paint and approximately the size of a grain of salt.And on the side of the tube under the hinge, place the CD NA pellet on ice and proceed immediately to cleaning the pellet. To begin the procedure for cleaning the CD NA pellet, use a P 1000 micro pipette to carefully remove all of the supernatant from the tube. Be careful not to disturb the pellet.It is your sample at 500 microliters of cold, 80%ethanol to the tube. Gently cap the tube and invert it slowly several times. Watch your pellet very closely to make sure it does not become detached from the side of the tube.If the pellet becomes detached, you can get it back to the bottom of the tube by either placing the tube back in the rack and letting the pellet settle to the bottom or centrifuging the tube at full speed for 15 seconds. Next, use a P 1000 micro pipette to carefully remove the ethanol without disturbing the pellet. Tip the tube to enable removal of as much liquid as possible.Add a new Eloqua of cold, 80%ethanol, cap the tube and invert it slowly several times. After removing all of the ethanol possible by using a P 1000 micro pipette. Centrifuge the tube at full speed for five seconds.Then use a P 10 micro pipette to remove the last few microliters of ethanol without disturbing the pellet. Place the open tube in a drying box for 10 to 20 minutes to evaporate the remaining ethanol. The pellet is easily lost once it is dry, so be careful to handle the tube gently and to close the cap.When drying is done, visualize the dried pellet to confirm it is present in the tube. Finally, resuspend the dried pellet in 22 microliters of RNA free water and place the tube on ice. This CDNA is then used to prepare biotin labeled CRNA by in vitro transcription using the Enzo BioArray kit.After the biotin labeled CRNA has been cleaned and quantified, it is fragmented for target preparation. Once the CDNA has been generated, we then use standard in vitro transcription methodology in order to generate biotinylated CRNA. The CRNA needs to go through fragmentation before it can be applied to the microray chip.To begin this procedure, transfer an amount equivalent to five micrograms of CRNA to a labeled 0.5 milliliter PCR tube. Add enough RNAs free water to bring the total volume to 16 microliters, and then add four microliters of five x fragmentation buffer to the tube. The total volume in the tube should be 20 microliters.Vortex the tube and centrifuge for 10 seconds. Then incubate the tube at 94 degrees Celsius for 30 minutes. In a thermocycler, put the tube on ice following the incubation.The fragmented and unfragmented CRNA from each sample is then assessed by electrophoresis using a 1.2%precast AROS gel. When the run is complete, the gel is visualized on a transluminator and an image is acquired. The final synthesis product is hybridized to the atric yeast gene chips and representative results from the microarray analysis are shown here.This first figure is an example of a scanned atrics yeast gene chip image generated by the atrics gene chip operating software. This is a 2D scatterplot of all genetic transcripts, about 6, 700 genes comparing control and treated yeast data. Each point represents a single gene.Genes colored in purple indicate genes that are differentially expressed while genes colored in red are not. In this example, most of the differentially expressed genes are those involved in cell cycle control as indicated by their descriptions. This flow chart from the database for annotation visualization and integrated discovery illustrates differentially expressed genes in an affected biological pathway.The genes indicated with a red star indicate the downregulated genes in the miotic pathway. Finally, representative results of a volcano plot generated using geo species gene sifter software are shown here. Control and treated samples were compared with a P-value cutoff of 0.05 and a 1.5 fold expression change cutoff.We chose using yeast because yeast are easy to grow quickly, but we also realized the most critical part of working with yeast is actually creating decent sphero plating. One of the things in microarray we don't want to do is actually do an incomplete digestion and actually only sphero plast cells that are in a G one or a G two M phase. And then you're doing a microarray experiment on yeast that are in a certain replication phase.Another complicated point that we try to bring home in this exercise is that pelleting CD NA people talk about every day, but it's not necessarily easy. And what we've done in our module is we've actually used specialized tubes and visualization reagents. So we can actually spin down DNA and you can visualize appellate, which makes it easier for students and teachers alike.So that can be used in all phases of DNA and RNA work After its development. This module has really paved the way for faculty at the Baccalaureate institutes to explore, possibly looking at other model organisms or other treatment regimens that may be compatible with existing curricula or possibly their own research interests. And to give you an example of some of the modifications that were made, there was one site that had looked at the effects of herbicide treatment on yeast or another that looked at LAMP IDE treatment in yeast, while another site looked at the developmental changes in a mammalian cell line that they were particularly interested in.And lastly, another site had looked at the impact of differential light sources on arabidopsis or plants. After you watch this video, you should have a good understanding of how to set up a microray experiment with undergraduate science students, including the isolation of total RNA from yeast, the generation of CDNA, and the fragmentation of biotin labeled CRNA. Hands-on experiences with such high level technologies helps to reinforce and strengthen undergraduate science education.

microarray-analysis-for-saccharomyces-cerevisiae

Research

JoVE Journal

Biology

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

A high-throughput method to globally study the organelle morphology in S. cerevisiae

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitive Genomic Screens of Barcoded Yeast Libraries

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A bottleneck in the ‘design-build-test’ cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

Learn microarray analysis using RNA extraction, cDNA synthesis, and bioinformatics in Saccharomyces cerevisiae. This computational method teaches gene expression profiling under oxidative stress to undergraduate students.

Microarray Analysis for Saccharomyces cerevisiae

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