Computational protocol: Control of the C. albicans Cell Wall Damage Response by Transcriptional Regulator Cas5

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Protocol publication

[…] Single colonies were inoculated into 3 ml of YPD and grown overnight at 30 °C. Each overnight culture was used to inoculate 200 ml of YPD to an OD600 of 0.1, which was then incubated at 30 °C with shaking for 2–3 doublings. At this point the cultures were divided into two 100-ml cultures. To one of the cultures, caspofungin (diluted in dH20) was added to a final concentration of 125 ng/ml. To the other culture an equal volume of dH20 was added. The cultures were then incubated with shaking at 30 °C for 1 h, at which point they were harvested by vacuum filtration and stored at −80 °C. RNA isolation, microarray analysis, and Northern analysis were performed as previously described []. Microarray slides were scanned using ScanArray Express microarray scanner (PerkinElmer, Wellesley, California, United States), and the signal intensities were extracted with GenePix Pro 4.1 software (Axon Instruments, Union City, California, United States). Raw signal intensities were corrected for dye labeling effects within and between all slides using the normalize.loess R-function [] implemented in an affy microarray analysis package []. The resulting data were imported into the Spotfire DecisionSite for Functional Genomics software suite (Spotfire, Somerville, Massachusetts, United States) and filtered according to the GenePix quality scores above 0 and signal-to-noise ratio above 2. The p-values for differentially expressed genes between the compared strains were subsequently calculated and further adjusted for type I error with Bonferroni's transformation using BioConductor multtest R-package [] (see The results of this analysis with adjusted p-values below 0.05 and absolute fold changes above 2 are listed in . All told, we compared three hybridizations with untreated wild-type cell samples and 12 hybridizations with drug-treated wild-type cell samples to identify caspofungin-responsive genes. We compared three hybridizations of drug-treated mutant cells to drug-treated wild-type cells to identify dependent genes for each mutant. RNA for each sample came from an independent culture. […]

Pipeline specifications

Software tools GenePix Pro, affy, multtest
Application Gene expression microarray analysis
Organisms Candida albicans, Saccharomyces cerevisiae
Chemicals Calcium, Zinc