Discussion

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            This experiment utilized microarray technology to analyze the entire genome of the Saccharomyces Cervisiae.  The data was collected under the two conditions known as wild-type and mutant.  The wild-type is exactly as it sounds and is considered to be the most common genotype and phenotype of the organism as it exists in the wild.  The mutant was a double knockout of both the ZMS1 and ZMS2 genes.  As these are genes involved in oxidative stress, there were 12 other genes analyzed using microarray technology (Table 1).  These genes were thought to be involved in oxidative stress as well.

            The comparison of both the wild-type and mutant microarrays would hopefully give some insight as to how the organism compensates for the loss of function of the ZMS1 and ZMS2 genes.  Unfortunately, no specific gene name could be found for the ZMS2 gene because there has not been much research on this gene to date.  Henceforth, the eleven remaining genes were analyzed.  This constituted one problem which occurred when attempting to analyze the data.  The ZMS1 gene is, on the other hand, well known and studied.  As seen in the results section, all of the eleven genes studied using MagicTool showed very similar expression patterns (Table 2).  Therefore, these genes are very likely closely related to one another both functionally and possibly evolutionarily.  This result is pictorially represented in Figure 4. 

            Some of the problems which were experienced while trying to perform the experiment were as follows: possible light exposure to the dyes, contaminated RNA by RNAses, and general experimental error as further described in the protocol section.  The dyes used, Cy5 and Cy3, are extremely light sensitive and will break down due to prolonged exposure.  The Cy5 dye (red) is notably more light sensitive and may be the leading reason as to why there is little/no red expression (Table 2, Figure 3).  Many precautions were taken to prevent this from occurring such as working in a nearly dark environment when handling them.  The tubes containing the dyes were always kept in the dark as well as being wrapped in aluminum foil for extra protection.  However, the kit form which the dyes were taken was not brand new.  The kit was within its expiration date, approximately one year old.  This may or may not have been a contributing factor to the dyes degrading but is a possibility which should be considered.

            RNA is by nature extremely fragile.  There were many precautions taken to ensure that none of the RNA sample was contaminated.  Everyone who came into contact with the sample RNA was sure to wash their hands, remove everything from the surface of the area being used, wiping down the area with RNA Zap, and being extra careful to avoid touching or even breathing in the direction of the sample.  However, even after all the precautions were taken, some of the RNA used for the experiment was degraded to the point that it was no longer usable.  This is reflected in the results section.  However, there was another lab group working on an identical experiment which was able to share their RNA allowing this experiment to continue.

            Also, whenever an experiment is being conducted there is always the chance of experimental or human error.  No one is perfect and inevitably there is the possibility of pipetting error or a miscalculation.  These are other factors which may have influenced the data which was collected.  Unfortunately, the only way to account for this type of error is to repeat the trials multiple times to ensure that the results obtained are consistent.

            As stated, this experiment could be improved by increasing the number of trials conducted.  Each group submitted only one slide to Davidson University for microarray analysis, and since this was the first time for each group performing the experiment, significant errors might have been made.  Making replicates of the slides would allow us to gain more familiarity with the procedure, possibly reducing errors in the procedure.  The experiment could further be continued by analyzing the genes.  To further continue the experiment, the site of the genes could be determined.  By knowing the exact chromosome in which the genes are located, the genes can further be analyzed to see if the location and function of them had much to do with the similarity in expression.