Results
Optical density at 600 nm was taken for the yeast cultures to be used for RNA extraction (Table 1). Yeast cultures were found to be in the mid-log phase (0.4-1.7 OD). The ideal range to extract RNA is the late-log phase (OD 1.5-2.5), however these strains are all that was available.
Table 1. Recorded values of Optical Density readings using a Spec-20 for 4 strains of Yeast cultures used in the microarray experiments.
| Yeast Samples | OD 600 |
| WT | 0.684 |
| Δzms1 | 0.654 |
| Δzms2 | 0.618 |
| Δzms1/Δzms2 | 0.606 |
Nanodrop analysis was done to determine the concentration of the extracted RNA (Table 2). Both samples also fell in the purity range of 1.9 - 2.2 (ratio of A260/A280), indicating the samples were of acceptable purity for probe construction.
Table 2. Nano drop readings of RNA samples extracted from Δzms2 and wild type Yeast strains.
| Yeast Strain | Conc. ng/uL | Purity (260nm/280nm) |
| WT | 227.3 | 2.17 |
| Δzms2 | 387.7 | 2.15 |
Gel Electrophoresis of RNA samples yielded obvious 28s and 18s rRNA bands as well as a band representing possible DNA contamination further up on the gel. A very faded band at the bottom of the gel showed possible presence of tRNA indicating that our sample was not completely purified. (Figure 1)
Figure 1. GelDoc picture of agarose gel electrophoresis of RNA extracted from yeast. Several groups shared this gel for analysis but the RNA samples used in this analysis are in lanes 5 and 6. Lane 5 contains the RNA sample for the WT and lane 6 contains the sample from the Δzms2 strain.
Using ScanAlyze, the microarrays were scanned and gridded for analysis (Figure 2). It was determined that a streak on the right hand side of the grid may cause erroneous data. Therefore, each spot was flagged to not be included in later analysis.
Figure 2. Grid pictures as seen in ScanAlyze. Pictures taken from Slide 725. Grid 11 is the Top Array. Grid 27 is the Bottom Array. The same genes are in the same location on both grids. The same grids were also analyzed by a different group from Slide 728.
When standardizing the top and bottom grids from slides 725 with the ones from slide 728, an over/under expression range of >2 and <-2 was chosen for selecting genes for further analysis. As the scale of the ratios is in a log2 transformation, a fold change of +2 reflects a +4x fold change in expression as our minimum selection value. It was important to note that in spite of standardization, the box-plot for column 2 (725 slide bottom grid) the mean expression value for this array was unequal to the other arrays. (Figure 3)
Figure 3.Box plot representing standardized array data from four arrays. Array 1 is Slide 725 Top (WT Red); Array 2 is Slide 725 Bottom (WT Red); Array 3 is Slide 728 Top (WT Green); Array 4 is Slide 728 Bottom (WT Green)
From the ScanAlyze data, a table was created using MagicTool. MagicTool also clustered the table, creating Figure 4. This table of ~200 genes was analyzed based on consistent expression across all arrays. Any gene which was consistently underexpressed or overexpressed in all arrays was selected for further analysis, which led to the list of genes in Figure 5.
Figure 4. Clustered view of entire set of arrays. Click on image for larger view of genes with corresponding expression values for each array. Column 1 contains gene names and columns 2-5 display expression values. Red expression values represent over-expression where green values represent under-expression.
These selected genes were chosen due to an overall consistency of expression between the arrays. Even though a given gene may not have had an expression value greater than 2 or less than -2 it may have been chosen if it surpassed the minimum expression level in a majority of the arrays.
Figure 5. Collection of 11 genes found to be have consistent trend for either up- or down-regulation.
Figure 6. Summary of biological process and molecular function of each of the 11 genes from Figure 4. Red represents a gene that is upregulated. Green represents a gene that is downregulated. Gene data was taken from the GCAT Yeast Genome Database.
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