Supplemental Materials & Methods

Microarrays: A Global Network Model of the Arsenic Response

I. Isolation of Total RMA from Yeast Treated with ASIII

1. Grow two 10ml yeast cultures during the day and check the cell number by hemocytometer at 4:00 PM. (Doubling time for yeast strain BY4741A is ~2.5 hours in synthetic complete medium).
2. Inoculate one large flask of 1300ml synthetic complete media with 10E4 yeast cells. Let it shake O/N at 30°C, 240RPM.
3. Count cells. Do not go over 10E7 cells (if into stationary phase then must inoculate another flask and let it shake during the day until it reaches no more than 10E7 cells). Split into 13 separate 100ml culture flasks. Treat as follows: Timepoints 0h, 0.5h, 2h, 4h – doses 0, 100uM Arsenic, and 1mM Arsenic. (Use Sodium Arsenite – 129.91g/mole and make a fresh stock solution in water to dilute from... Can store stocks at -80°C for no more than two weeks).
4. Spin cultures (3500 RPM 5 minutes) in 50 ml conical tubes, wash with 25ml water, combine, spin again.
5. Quick freeze pellet in dry ice and store at -80°C or go on to RNA isolation.
6. Resuspend pellet in 4 ml lysis buffer (10mM Tris-HCL pH 7.5, 10mM EDTA, 0.5% SDS.
7. Add 4 ml acid (water saturated, low pH) phenol. Vortexed well.
8. Incubate at 65°C for 1 hour with occasional vigorous vortexing.
9. Place on ice 10 minutes, centrifuge at 4°C for 10 minutes.
10. Remove aqueous layer, re-extract with Phenol (room temp., no incubation).
11. Extract once with chloroform.
12. Add sodium acetate to 0.3 M + 2 vols. Ethanol, -20°C for 30 min., spin, wash pellet 2-3 times with 70% ethanol.
13. Quantitate by OD 260-280 and run on gel.
14. Follow up with Qiagen Poly-A isolation.

Note: Since changing to the Agilent platform, we have been isolating total RNA with the Qiagen RNEasy enzymatic (zymolase) protocol. No Poly-A isolation is needed.

 Back to top

II. A. Explanation of Specific Deletion Experiment Signature Gene Lists

1. Intensity plots were generated from each experiment in Resolver. A gene was considered a signature gene if the p value was less than 0.001 and if the fold change value was greater than or equal to 2 fold.
2. Signature genes were broadcasted on the intensity plot and exported as text files.

II. B. Explanation of Resolver "Minus" Lists (Control vs. Arsenic Treated)

1. Signature gene lists were generated in Resolver from intensity plots as described above in A1.
2. Each signature gene list was saved as a "Bioset" in Resolver. The parent Bioset was compared to each Deletion Bioset using the "Minus" function. This function finds those members in Bioset Group 1 (parent) that do not exist in Bioset Group 2 (deletion). Each of the resulting lists was saved as a new Bioset.
3. The new "minus" Bioset was broadcasted on its corresponding intensity plot and exported as a text file. This was repeated for each experiment.
4. The lists were analyzed in GeneSpring. Genes were eliminated from the list if they showed any kind of non-significant expression. In other words, if a gene did not exist in bioset 2 because it did not meet the significance criteria but the profile still showed it to be differentially expressed, it was eliminated.

II. C. Explanation of GeneSpring Filter on Fold Change Comparisons (Control vs. Arsenic Treated)

1. Signature gene lists were generated and saved as described above in A.
2. Lists were imported into GeneSpring. Each Deletion signature gene list was combined with the Parent signature gene list and saved as a new list.
3. The "Filter on Fold Change" function was used to compare the Parent control vs. Parent AsIII experiment with each deletion (AsIII) experiment. The gene list selected for each Filter on Fold Change analysis was a combination of the Parent signature gene list and the signature gene list of the AsIII Deletion being analyzed at the time. For example, if the comparison was being done between Parent AsIII and Yap1 AsIII, the list used in the analysis was the combination of the parent signature genes and the Yap1 signature genes. The Filter on Fold Change function reports genes were selected from the one condition (Parent) that had Normalized Data values that were greater or less than those in the other condition (Deletion under investigation) by a factor of 2 fold. Each resulting gene list was saved.
4. All of the resulting gene lists were combined and an annotated gene list was exported for use in Eisen's Cluster/Treeview package. The format of the exported data was the natural log. The gene tree generated for the paper was generated in GeneSpring.
5. Each Filter on Fold Change was saved as an annotated gene list. Additionally, a combination of all Filter on Fold Change lists was saved.

 Back to top