|
| Status |
Public on Jan 04, 2010 |
| Title |
YPD_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Log-phase culture grown in YPD
|
| Organism |
Candida albicans |
| Characteristics |
agent/stress: none
genotype/variation: SC5314 (wild-type)
|
| Treatment protocol |
In the second (H2O2) experiment, RNA was analyzed from H2O2-exposed cells. In this case, the final O.D. 600nm ranged from 0.5-0.9 before exposure to 0.4 mM H2O2 in YPD for 10 minutes at 30°C. The cells were immediately centrifuged into a pellet and flash frozen with liquid nitrogen for storage. RNA was then prepared and analyzed as above.
|
| Growth protocol |
In the first (YPD) experiment, wild-type and rtt109 -/- cultures were grown at 30°C in 50 ml YPD to a matching O.D. 600 nm for each of the four biological replicates, with the final O.D. 600 nm ranging from 0.6-0.9 for the different experiments. Pellets were flash frozen with liquid nitrogen and stored at -80°C until further processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified by acid-phenol extraction followed by ethanol, ammonium acetate precipitation as previously described (Sexton et al., Yeast 24, 847 (2007))
|
| Label |
Cy5
|
| Label protocol |
10 μg RNA was labeled by reverse transcription using the 3DNA Array 350 system (Genisphere).
|
| |
|
| Channel 2 |
| Source name |
Log-phase culture grown in YPD
|
| Organism |
Candida albicans |
| Characteristics |
agent/stress: none
genotype/variation: PKA13 (rtt109-/-)
|
| Treatment protocol |
In the second (H2O2) experiment, RNA was analyzed from H2O2-exposed cells. In this case, the final O.D. 600nm ranged from 0.5-0.9 before exposure to 0.4 mM H2O2 in YPD for 10 minutes at 30°C. The cells were immediately centrifuged into a pellet and flash frozen with liquid nitrogen for storage. RNA was then prepared and analyzed as above.
|
| Growth protocol |
In the first (YPD) experiment, wild-type and rtt109 -/- cultures were grown at 30°C in 50 ml YPD to a matching O.D. 600 nm for each of the four biological replicates, with the final O.D. 600 nm ranging from 0.6-0.9 for the different experiments. Pellets were flash frozen with liquid nitrogen and stored at -80°C until further processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified by acid-phenol extraction followed by ethanol, ammonium acetate precipitation as previously described (Sexton et al., Yeast 24, 847 (2007))
|
| Label |
Cy3
|
| Label protocol |
10 μg RNA was labeled by reverse transcription using the 3DNA Array 350 system (Genisphere).
|
| |
|
| |
| Hybridization protocol |
DNA oligonucleotide C. albicans microarray slides were purchased from The Genome Center at Washington University (http://genome.wustl.edu/services/microarray/candida_albicans). Hybridization and washing were performed per the labeling manufacturer’s instructions (Genisphere).
|
| Scan protocol |
Scanning was performed immediately after washing using GenePix software at a PTM ranging from 490-630 (Cy3; 532nm) and 650-960 (Cy5; 635nm).
|
| Description |
Analysis compared steady-state RNA levels of log-phase wt and rtt109-/- cells
|
| Data processing |
Limma package (limma_2.18.0) from Bioconductor was used for preprocessing and model fitting. A linear model was fit to the background-corrected, loess-normalized and log-transformed expression data. The dye effect and mutant effect were tested as explanatory variables to determine whether the expression level of each gene differed between mutant and wild type samples, after the dye effect was removed statistically. Three features for each gene were printed on the array, resulting in three estimated log-fold changes for each gene. Genes with at least one feature having a p-value < 0.01 for mutant effect and a fold change ≥2 were considered potentially differentially expressed in mutant and were included for subsequent Gene Ontology (GO) enrichment test. The gene and GO term association list was downloaded from http://www.candidagenome.org (version 1.633 and taxon: 5476) with duplicates removed. All the ancestor GO terms were added to the association list using GO.db package (GO.db_2.2.11) from Bioconductor. GO enrichment analysis was performed using the hypergeometric test in R (V 2.9.0). GO terms with at least ten associated genes in the genome and a p-value adjusted using the Benjamini-Hochberg method < 0.01 were considered significantly enriched.
|
| |
|
| Submission date |
Nov 09, 2009 |
| Last update date |
Dec 29, 2009 |
| Contact name |
Jessica Lopes da Rosa-Spiegler |
| E-mail(s) |
jessica.lopesdarosa@umassmed.edu
|
| Organization name |
University of Massachusetts Medical School
|
| Street address |
364 Plantation street
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL9545 |
| Series (1) |
| GSE18936 |
RNA abundance in wild-type and rtt109 -/- Candida albicans, with and without peroxide stress |
|
