Discussion: 

    The overall goal of this experiment was to use microarray analysis to look at the changes in expression levels of yeast cells with genes that had mutations in a pathway that dealt with oxidative stress. Dr. Slekar identified two genes that effected this pathway, ZMS1 and ZMS2, and provided the yeast with these mutations. The goal of this experiment was to use microarray analysis to look at the differences in the expression profile between ΔZMS1 and wild-type ZMS1 yeast.  It was hypothesized that the levels of gene expression for the ΔZMS1 mutant would be lower than in the wild-type yeast.

    Table 2 in the results section shows the purity of the RNA isolated from Dr. Slekar's wild-type and mutant yeast cells. The optimal purity range for the A260/A280 calculation is between 1.8 and 2.0. From looking at the purities in Table 2, both samples were in between the range of 1.8 and 2.0, meaning both samples had good purity for nucleic acids. Based on this alone it could not be concluded that the samples contained pure RNA. Figure 1, showed the results of the samples that were run on a 1.2% agarose gel. Lane 6 shows a large band of DNA which indicated that the wild-type sample did not contain pure RNA despite the high purity reading. This is because the purity reading can only select for nucleic acids, which includes both DNA and RNA. This also would indicate why the concentration of the wild-type sample was so high compared to the ΔZMS1 sample and other samples from the class. It should be noted that the wild-type sample with the high DNA concentration was not used in the experiment, so errors in the microarray slide could not be accounted for by this.

    Results of the microarray slide (13697727) showed very minimal data. This may have been because of errors in the procedure. During the wash/hybridize stages of the microarray preparation difficulties were encountered with applying the cover slip on the slide. Due to this issue the cover slip was not correctly placed on the first attempted leading to it being removed and repositioned. This could have lead to a smearing or detaching of the genes attached to the slide. Also in the washing/hybridization steps the solutions that were needed to wash the slides may have been incorrectly diluted. Incorrect dilutions would effect the strength of the solutions and therefore their ability to wash the slides. Another issue with the wash/hybridization stage may have been with the sharing of the wash solutions with other groups. Washing with used solutions may have effected the ability of the solution to wash the slide. Other possible reasons for the minimal data that was received could have been, the cDNA did not hybridize to the oligo nucleotides attached to the slide and were subsequently washed away during the wash stages. Also there could have been a degradation of the Cy-5 (red) dye because when the slide was examined after being scanned the only visible spots were green. The same dye degradation could have also effected slide 104 from 2004, where the data used in the results section was obtained from. This may explain why most of the genes seem to be under expressed, because not many red spots were seen on slide 104. Further issues with slide 104 could have come from the gridding technique used in Scanalyze. When adjusting the spots in the grid to fit the spots on the slide some grid spots may have been to large or to small for the slide spots within them.

    Looking at the problems that were encountered in the procedure this experiment could be improved by using different dyes that do not have degradation issues, so the data could be visualized. This experiment could be further improved by using different methods for washing/hybridizing the slides such changing the orientation of the slides during the wash stage, meaning keeping the slides horizontal while being washed and using another container besides a Coplin jar. Having more of the solutions available would decrease the inconsistencies caused by different dilution techniques. Another way to improve the experiment would be to have more microarray slides available for both practicing techniques and to increase the amount of data generated.

    For this experiment to be continued, the first step would be to redo the microarray portion of the experiment to generate the necessary data that was not originally obtained. Once this is completed further analysis of the genes in the oxidative stress pathway that involves ZMS1 could be examined. An example of this would be a more in depth look at the Pho85 related genes and thus the relationship between ZMS1 and Pho85. The cursory analysis of the Pho85 genes done in the results of this experiment would be a starting point for this further analysis.

    The results of this experiment were inconclusive. This is because the data that was obtained from slide 104 was not useful in showing any relationships between the genes. Figures 4,5, and 6 all show the inconsistencies between the top and bottom of the slide making it difficult to make any conclusions about the genes. Some Pho85 related genes were examined to try and determine the effect of the ΔZMS1 on the hypothesized pathway presented for this experiment. Figure 4 shows why its difficult to make any conclusions about the effect of this mutation since the top and bottom show very different expression ratios. Due to the fact that the original slide for this microarray yielded no usable data and the slide provided from 2004 did not have consistent data the hypothesis could not be supported or rejected. In order to draw conclusions about the hypothesis the experiment would have to be repeated.