Discussion
The purpose of this experiment was to learn how to use and analyze a microarray. The yeast cells provided for the group by Dr. Slekar were mutant cells missing the ZMS2 gene. The experiment proceeded well from the beginning with the concentrations of RNA from both the mutant and wild-type yeast cells being on the higher side of the expected range (Fig. 2). When the gel of the RNA was run the bands were distinct, not smeared which would signify RNA degradation (Fig. 3). However, the ratios of the A260/A280 were right outside of the range for purity with the wild-type at 2.35 and the mutant ∆ZMS2 at 1.83 and the ratio range being 1.9-2.2.
As noted in the methods we unfortunately were unable to use our own data from the experiment and instead replaced our slide with one from a previous year which was actually a ∆ZMS1/∆ZMS2 knockout instead of a ∆ZMS2. The main difficulties with our slide were that the colors on the slide were very faint, in some regions there were smears of green dye that interfered with the color on the microarray spots, and that the red signal was relatively unseen. These problems could have been due to multiple reasons including an ineffective wash, the use of a slightly off incubation temperature (could lead to a poor hybridization), inexact pipetting of the two dyes (ie. not enough of one dye or too much of another or both), or even the presence of too much light (due to someone accidentally walking into the room while we were working). Also the cDNAs might not have been made correctly (impure RNA) or the RNA could have been affected by RNAses or inhibitors.
When conducting the procedures in the experiment the group did not encounter any known problems. However, since there were four people working on the same project there is always the chance of mistakes. After the relative concentration of RNA in the cell sample had been determined and the RNA was isolated and run on a gel, the need to eliminate the presence of RNAses was critical in the outcome of the lab. Since RNAses are found on the skin and hair, keeping the work area free of them was difficult. If any one of the four members happened to have touched her hair or face while working on the lab she could have damaged the RNA without realizing it. Group miscommunication could also have been a problem in carrying out the experiment. Some of the work had to be completed outside of the normal lab period which left the group members completing different portions of the experiment alone, and if some detail had been overlooked in one of the washes or if the amount of light in the room because of the time of day had degraded the dye the microarray slide may have come out streaked or with poor dye signals as our slide did.
The next time the lab is performed it would be better if the group members were able to meet to perform each step of the experiment together. That way they could check each other to make sure that, for example, if one touches her hair that she then changes her gloves, or that someone else entering the room does not turn on the light, or even to make sure that some step of the protocol is not overlooked. Since the data we collected was not useful we used slide #106 from a group who performed the experiment in the fall of 2004. However, when the data from that slide was analyzed we found no useful information because, of the 6 genes we found to be the most down-regulated (Figs. 7 and 8), none of them had any correlation to the oxidative stress relief process that the deleted genes – ΔZMS1 and ΔZMS2 – helped to control. This was not entirely unexpected because, as discussed above, the microarray process has not been perfected and there are many opportunities to make mistakes. The slide from the 2004 group was an improvement over our original one and allowed us to complete the analysis process, but it is still subject to error. We do not know what kind of mistakes the 2004 group may have made, but it is likely that they did make a few. Thus, the data we worked with was decent, but far from perfect.