Strain: JM43 (wild-type), Medium: SSG-TEA, Condition: a pool of RNA collected from both anaerobic and aerobic cultures
Extracted molecule
total RNA
Label
Cy5
Description
Sparged, fermentor cultures of a wild-type yeast strain (JM43) were aerobically grown on galactose medium (SSG-TEA) in the presence of Antimycin A for three generations. After three generations, the sparge gas was switched from air to O2-free N2 and samples were harvested after 0 (aerobic control), 0.04, 0.08, 0.13, 0.19, 0.25, 0.38, 0.5, 1, 2, and 3 generations of anaerobic growth in the presence of Antimycin A.. Total RNA was reverse transcribed (Cy3) and hybridized against a reference (Cy5).
Data processing
GenePix Pro software (v4.1) was used for spot identification and fluorescence intensity quantification. After manually flagging and removing spots with aberrant measurements, background fluorescence was subtracted from the median Cy3 and median Cy5 fluorescence intensity values. Any resulting negative intensity values were set to zero and a constant of one fluorescent unit was then added to all intensity values. Outliers were identified and removed using SAS, and the log2Cy3 intensity (query cDNA) for all remaining observations on a slide was normalized against that of the log2Cy5 intensity (reference cDNA) using locally weighted linear regression (Loess). The log2(Cy3/Cy5) ratio for each spot was calculated, and the mean log2(Cy3/Cy5) ratio across all observations on a slide was normalized to a value of zero. The mean of the normalized log2(Cy3/Cy5) ratio for each gene was then calculated by averaging the duplicate observations on each slide and pooling replicate slides by sampling time.
