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RNA Isolation and Analysis - Labeling cDNA
The OD readings for our yeast cultures were of values of zero and awkwardly negative numbers therefore the cultures could not be used as viable sources of RNA; no RNA gel is therefore available. Samples from another group were used to accommodate this problem.
Hybridization
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Figure 1. A pictorial example of a hybridization slide used to conduct this microarray analysis* |
Microarray Data
| Figure 2. One grid section of the many that comprised the final micorarray image. There is mild background interference seen on the image; however, light but distinct expression dots can be feasibly seen. Each grid measured 24 columns by 22 rows. |
A list of approximately 12 genes were researched based on their involvement in the yeast organisms responses to oxidative stress or their role in normalizing the cell when subjected to varying oxygen environments. ZMS1 and ZMS2 genes are among the 12 - refer to the below Table 2 for further description.
| GENE | ALT. NAME | DESCRIPTION |
| YPL061W | ALD6 | aldehyde dehydrogenase activity |
| YNL241C | ZWF1 | involved in adapting to oxidative stress conditions |
| YJR127C | ZMS1 | involved in transcripton control of nuclear and mitochondria genes |
| [NONE] | ZMS2 | not a lot of research information on; still being explored |
| YDL033W | GDP1 | essential for growth under osmotic stress |
| YAR007C | RFA1 | highly conserved single stranded binding protein |
| YPR031L | PHO85 | involved in environmental stress response |
| YDL059W | GDP2 | expression controlled by O2-independent signaling pathway |
| YLR006C | SSK1 | mediates response to oxidative stress |
| YGR097W | ASK10 | component of the RNA polymerase II holoenzyme that is phosphorylated in response to oxidative stress |
| YMR250W | GAD1 | required for normal oxidative stress tolerance |
| YPL202C | AFT2 | required for iron homeostasis and resistance to oxidative stress |
| Table 1. A brief however comprehensive display of 12 genes of interest in the oxidative stress pathway along with ZMS1 and ZMS2 genes, which were the double-knockout mutants in this microarray investigation. This list was used as a reference for deciphering expression patterns of the double-knockout mutant yeast. |
It was one of the goals of this experiment to determine if gene expression patterns changed on account of the double-knockout mutation. The selected genes as displayed in Table 1 were referenced for potential changes and the subsequent Table 3 displays the expression values whereas the below Figure 3 and Figure 4 illustrates the correlations.
| Table 2. This table is representative of the above Table 1 gene expression values. As can be seen by the slots labeled "missing," a few of the genes did not show viable expression ratios which can be attributed to the fact that there was an unequal distribution of green and red expression; there was hardly any red expression. |
| Figure 3. Plot analysis conducted on the highlighted 12 genes of interest, as displayed in Table 2. Several genes lacked overall expression, thus the plot reflects a more narrow representation of the expression similarities of the genes. Clustering of this data using MagicTool software yielded further confirmation that these genes are very closely related in expression levels as seen by their relative closeness on the tree produced. Unfortunately this tree could not be reproduced to display due to complications with the software program. |
| Figure 4. Circular diagram representing the similarities that each gene of interest has to the others based on calculated expression values. Despite the lack of image clarity, it can be noted that each gene demonstrated very similar expression values therefore are closely related to one another as seen in the multiple connections each one makes. |
*Picture from : http://www.transcriptome.ens.fr