Index        Introduction        Results        Discussion        References        Individual Project - Karl

Materials and Methods:

    During the first two weeks of our experiment we isolated RNA from mutant yeast (RNA Isolation from Yeast) to label our microarray.  There were five steps that we took, growing the yeast cultures, preparing spheroplasts, determining the concentration of our yeast RNA, and checking the RNA for degradation.  This procedure, and all the procedures listed below, are outlined in the Bio480/580 lab manual, available if you click the brown links.

    Yeast cultures of S. cerevisiae obtained from Dr. Slekar’s lab were cultured in 50ml of media in 250ml flasks.  Cells of the Wild Type and ZMS1 mutant were inoculated into media under high sterile condition and grown overnight in a shaking incubator at 30^oC overnight.  The yeast cells were harvested after 24hrs.  The growth phase was determined based on optical density recorded with spectrophotometer.  The spectrophotometer was set to a wavelength of 600nm and readings were expected to be between 1.5-2.5.  Using the chart in this protocol, the RNA quantity we intended to use was estimated.

     Yeast cell sphaeroplasts were prepared by digesting cell walls of yeast with enzymes. Cells were centrifuged at 2,500 RPM (1,500 x G) for 5min and pellets were resuspended with 1ml of Potassium Phosphate/Sorbitol buffer (1.2 M sorbitol, 10 mM potassium phosphate, pH = 7.2).  3µl b mercaptoethanol and 320 µl of 1 mg/ml lyticase were added to degrade cell wall components.  Another wash was repeated with of 500µl Potassium Phosphate/Sorbitol buffer, spurned and set on ice. See manual for full protocol.

    RNA isolation was carried out with the RNAsafe kit (Qbiogene).  Pellets of sphaeroplast were resuspended in 800 µl of solution and RNA isolation was done with the RNAsafe kit (Qbiogene).  200 µl each, of solution 1 and 2 were used to inactivate cellular RNases released after cell lyses as well as to help stabilize the RNA in solution.  800 µl of isopropanol was used to precipitate RNA.  RNA pellets were then resuspended in 100 µl of Solution 3.  20 µl of solution 4 was used to bind DNA.  The tubes were incubated for 5mins at room temperature.  Supernatants were collected and stored in the refrigerator at -4^oC.

    2 µl of RNA was quantified using the nanodrop device (Thermo Fisher). Readings were taken at the A260 wavelength and RNA purity was determined by the A260/A280 ratio.  A ratio of 2.1 would be considered pure.  RNA extracts were run on a 1.2% Agarose gel for 30minutes at 100volts to identify degradation if any.   In addition, 1 µl of ethidium bromide was added to each tube. Bands were visualized and a photograph taken.  

    RNA samples were prepared for the probe (Labelling the Probe) for our microarray analysis using the Genisphere 3DNA Array 350 Detection Kit.  During the first step we reverse transcribed our mRNA into cDNA using a specialized primer.  In the second step we labeled the ZMS1 with the red dye (Cy 5) and the Wild type Green (Cy 3).  The table below shows the amounts which were mixed to a total volume of 14 µl for our probe.

     6 µl of the master reaction mix was added to each tube above and incubated at 42^oC for 2hrs after which the reaction was stopped by adding 3.5 µl of 0.5 M NaoH / 50 mM EDTA to denature and degrade RNA hybrids. The resulting solution was neutralized with 5 µl of 1 M Tris-HCl, pH 7.5 to bring it to a total volume of 29.5 µl.  Both products mixed in one tube and brought up to a final volume of 130 µl of cDNA.  A YM30 micro concentrator was used to concentrate the cDNA, it was first rinsed with 100µl of TE buffer and then 130 µl of the cDNA sample was passed through the membranes, spun and then eluted with 5 µl of TE buffer.

    The micro-array slide, with  barcode no: 13760694, was prehybridized (Hybridization of the cDNA Probe) in Coplin jar with 50 ml of 3X SSC, 0.1% SDS, 0.1 mg/ml sonicated salmon DNA.  The hybridization solution was made up according to the amount indicated in the table below to a total volume of 58µl:

    The slide was incubated for 10minutes at 80^oC and 42^oC. All hybridization solution made was poured onto the slide taking care to spread the solution evenly. Cover slip was carefully placed on the slide to avoid air bubbles forming.  Slide was transferred into a 50ml tube, sealed with paraffin and incubated at 37^oC for 24hrs.  The slide was washed at 15mins intervals in 2x SSC + 0.2% SDS (incubated at 55^oC), 2X SSC and 0.2X SSC while shaking gently.  The second hybridization, a light sensitive process, was done under minimum light.  A hybridization mix was prepared and transferred onto microarray slide.  A cover slip was placed carefully upon the slide, which was transferred into 50ml tubes wrapped with foil, , sealed with paraffin to prevent dehydration, and incubated at 37^oC overnight.  A serial wash was repeated as explained earlier in the same sequence, and the microarray slide was then dried and cleaned for packaging.  Tubes with the slides were transferred into Styrofoam packaging and sent out  to Dr. Cambell at Davidson College for scanning. 

    Microarray pictures were analyzed with the MAGIC TooL software.   Background intensities were subtracted from gene expression intensities, the logarithm to the base two was taken, and the data was standardized in order that it would be better analyzed, to reduce errors due to background noise, impurities, or dye bias.

Noble Egekwu - egekwuni@jmu.edu    Karl Gorzelnik - gorzelkv@jmu.edu    Jonathan Baugher - baughejl@jmu.edu