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.
