METHODS
Total RNA Isolation from yeast:
In this first step, ZMS1Δ mutants and wild type yeast cells were cultured and made into spheroplasts. From the vulnerable spheroplasts, all RNA from the cells was isolated and quantified using spectrophotometry. The RNA was then checked for degradation using gel electrophoresis.
Isolated mutant and wild type RNA were separately reverse transcribed into cDNA for each respective sample using a special dye capture primer, and then concentrated down to better work with during later hybridization protocols. Note that in Step 6 of the reverse transcription method in the above link used 4ul of 5X RT buffer, 1ul of 10mM dNTP's, 1ul of Superase In, and 1ul of Reverse Transcriptase instead of the cocktail solution listed.
Protocol for this procedure was divided into the following six steps that involved the preparation of the TR2129 microarray slide and the cDNA:
1. Preparation of the DNA chips, which involved the removal of excess salt as well as the denaturizing of bound oligos.
2. Pre-Hybridization, which involved the preparation of the genome slide for the binding of the yeast cDNA. Note that in step five, the slide was put in a clean 50 mL tube then placed in a 55oC incubator.
3. Hybridization, which was the application of cDNA to the gene slide and subsequent binding.
4. Washing to remove the excess cDNA and possible contaminants.
5. Dye Marking, the hybridized cDNA was tagged with photosensitive dye, the ZMS1Δ mutant was dyed green and the wild type was dyed red.
6. Final washing to remove excess dye, which went awry by initially using a 2X SSC mixture without SDS.
The microarray was sent to Dr. Cambell for analysis at Davidson College.
Data Processing:
After the microarray returned from Davidson college, the data received was analyzed using Scanalyze and Microsoft Excel. In Scanalyze, both trials on the chip were segmented and a grid was added so the pixel target areas were appropriately over the correct gene spots. A god list was used to designate what gene the each spot in the grid contained. The pixel count for the gene spots were then calculated and exported to an Excel spreadsheet. From the Microsoft Excel spreadsheet obtained, in collaboration with a group doing a dye reversal of the same experiment, expression ratios were initially calculated by dividing the pixel number of the mutant color over the wild type pixel color. The ratios were checked for accuracy and viability by performing a global normalization. The global normalization was done by making a wild type pixel vs. mutant pixel graphs using the data collected for the Green ZMS1Δ mutant and comparing it to the red wild type and green wild type pixel counts (Figure 3. and Figure 4.) . After normalizing, a background check was done for the green mutant microarray by taking the average empty gene spot pixel count and adding two standard deviations. By performing a global normalization and background check for the green ZMS1Δ mutant microarray, genes were checked for by using approximately three fold up or down regulation. The genes seen to show a change in regulation were then compared to the other ZMS1Δ microarray group as well as the cross data, which was the green mutant and green wild type ratio, in order to see if the gene pattern noted was similar across groups.
Title Page Introduction Results Discussion References
Contact Information:
Josh Krueger Kris Tillett Gordo McGuire