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            Optical densities of the yeast strains were obtained (Table 1).   Optical density can be used to determine the stage of growth for each yeast strain.  The optical densities were similar for each strain, which suggests that the yeast cultures were in the mid-log growth stage.

Table1.  Optical densities of each yeast strain were obtained.  The optical densities were similar for each strain and suggest that the yeast cultures were in the mid-log growth stage.

            Total RNA was extracted from wt and Δzms1Δzms2 yeast strain.  Concentrations (at 260nm) and purities were determined through a nanodrop (Table 2).  Pure RNA has purity values (A260nm/A280nm) between 1.9 and 2.2.  Our purity values of 2.165 and 2.15 are within this range.  The total RNA extracted from the wt and Δzms1Δzms2 yeast strains was run on an 1.2% agarose gel to assess possible degradation (Figure 1).  The 28S and 18S bands were present for both the wt and Δzms1Δzms2 RNA samples.  The 28S and 18S bands were fainter on the wt RNA, but were clearly visible with careful inspection.  However, the Δzms1Δzms2 total RNA may have had some DNA contamination.

Table 2.  Concentrations and purities were determined for the RNA extracted from the wt and Δzms1Δzms2 yeast strains.  RNA with a purity value between 1.9 and 2.2 is generally considered pure.

Figure 1.  The total RNA extracted from the wt and Δzms1Δzms2 yeast strains was run on an 1.2% agarose gel.  Lane one contained wt total RNA while Lane two contained Δzms1Δzms2 total RNA.  Lanes three and four were used by another laboratory group.  The wt and Δzms1Δzms2 total RNA had bands at 28S and 18S, indicating the presence of un-degraded tRNA.  The Δzms1Δzms2 total RNA may have had some DNA contamination.

            The microarrays were scanned at Davison College and loaded into a TIFF file.  The TIFF files was opened via Scanalyze and grids were applied (Figure 2).  Spots with questionable data were flagged and not used in the analysis.  Grids 5 and 6 were analyzed from slides 13760722, 13760695, and 13760694 and grids 21 and 22 were also analyzed on slide 13760722.  Slides 13760695 and 13760694 labeled the wt with Cy5 (red) and Δzms2 with Cy3 (green).  Slide 13760722 had the dyes reversed, with wt labeled with Cy3 (green) and Δzms2 labeled with Cy5 (red).  The Cy3 and Cy5 dye intensities were plotted against each other to assess dye bias on the microarray slides to be analyzed (Figure 3). The slope of the best fit line for the dye intensity plots was 5.0935, .01, and .2378 for slides 13760722, 13760695, and 13760694 respectively.  A theoretical microarray with no dye bias would have a slope of 1.  The background intensity was subtracted from the foreground intensity and ratios of mutant over wild type were calculated.  A log base 2 transformation was applied to the ratios in magic tool (Figure 4).  Further normalization was performed via magic tool by setting the mean to 0 (Figure 4).  This normalization produced a more normal distribution for analysis.  Box plots created via Magic Tool showed that the different microarrays had similar means and distributions after normalization (Figure 5).

Figure 2.  This picture represents the top 16 grids of microarray slide 13760721.  The grids were added and adjusted via Scanalyze.

Figure 3.  These graphs represent the green and red intensities from each of the Δzms1 microarrays.  Best fit lines were applied to each data set.  Graph 1 has a slope of 5.0935 and represents slide 13760722 with Δzms1 labeled with Cy5 and wild type labeled with Cy3.  Graph 2 has a slope of .01 and represents slide 13760695 with Δzms1 labeled with Cy5 and wild type labeled with Cy3.  Graph 3 has a slope of .2378 and represents slide 13760694 with Δzms1 labeled with Cy3 and wild type labeled with Cy5.

Figure 4.  Histograms of the dye intensity ratios before and after normalization are displayed.  The ratios of mutant/wt underwent a log base 2 normalization to correct for the right skewed data (left).  Normalization moved the mean closer to zero.

Figure 5.  Box plots were created via Magic Tool.  The plot on the left represents the four microarrays before mean normalization, while the plot on the right represents the four microarrays after mean normalization. The box plot with mean normalized data displayed similar means and distributions between the different microarrays.  The columns correspond to the different microarray trials:  Column 1 (Δzms1 stained red on top grids of slide # 13760695), column 2 (Δzms1 stained red on top grids of slide # 13760722), column 3 (Δzms1 stained green on top grids of slide # 13760694), and column 4 (Δzms1 stained green on bottom grids of slide # 13760694).

            Genes in grids 5 and 6 were analyzed in Magic tool to determine which genes are differentially expressed in Δzms1 yeast.  Genes were considered differentially expressed if there was a three-fold increase or decrease in 3 out of the four array trials.  Twelve genes in grids 5 and 6 fit these criteria (Table 3).  Differentially expressed genes were compared to the yeast genome from the GCAT website (Table 4).

Table 3.  Genes that are differentially expressed in grids 5 and 6 in Δzms1 yeast are displayed in the Magic Tool format.  Genes were considered differentially expressed if the gene had a three-fold increase or decrease in three of the four array trials.

  Table 4.  The genes that were considered differentially expressed were compared to the yeast genome gene list available on the GCAT website.  Red color corresponds to upregulated genes, while green color corresponds to downregulated genes in Δzms1 yeast.