|
| Status |
Public on Jul 02, 2013 |
| Title |
WT_untreated_0hr_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
WT, untreated, 0hr
|
| Organism |
Candida albicans |
| Characteristics |
treatment: untreated
incubation time: 0 hours
genotype: WT
|
| Treatment protocol |
These overnight cultures were then diluted to an initial optical density (OD600) of 0.1 in YPD broth (50 ml) and then grown at 30°C until logarithmic growth phase and then treated with H2O2.
|
| Growth protocol |
Overnight cultures of C. albicans strains WT (SRR1/SRR1), srr1Δ/Δ (srr1/srr1), and gene reconstituted strain (srr1Δ/Δ+SRR1) were grown in YPD broth at 30 degrees C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cells by the hot phenol method.
|
| Label |
biotin
|
| Label protocol |
First and second strand cDNA was synthesized from 15 μg total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and oligo-dT24-T7 primer (PrOligo) according to the manufacturer's instructions. cRNA was synthesized and labeled with biotinylated UTP and CTP by in vitro transcription using the T7 promoter-coupled double-stranded cDNA as template and the Bioarray HighYield RNA Transcript Labeling Kit (ENZO Diagnostics). Double-stranded cDNA synthesized from the previous steps was washed twice with 70% ethanol and suspended in 22 μl of RNase-free water. The cDNA was incubated as recommended with reaction buffer, biotin-labeled ribonucleotides, dithtiothreitol, RNase inhibitor mix, and T7 RNA polymerase for 5 h at 37 °C. The labeled cRNA was separated from unincorporated ribonucleotides by passing through a CHROMA SPIN-100 column (Clontech) and ethanol precipitated at −20 °C overnight.
|
| |
|
| Hybridization protocol |
The cRNA pellet was suspended in 10 μl of RNase-free water and 10 μg was fragmented by ion-mediated hydrolysis at 95 °C for 35 min in 200 mM Tris-acetate (pH 8.1), 500 mM potassium acetate, 150 mM magnesium acetate. The fragmented cRNA was hybridized for 16 h at 45 °C to the C. albicansNimbleExpress GeneChip arrays. Arrays were washed at 25 °C with 6 × SSPE, 0.01% Tween 20 followed by a stringent wash at 50 °C with 100 mM MES, 0.1 M NaCl, 0.01% Tween 20. Hybridizations and washes employed the Affymetrix Fluidics Station 450 using their standard EukGE-WS2v5 protocol. The arrays were then stained with phycoerythrein-conjugated streptavidin (Molecular Probes).
|
| Scan protocol |
The fluorescence intensities were determined using the GCS 3000 high-resolution confocal laser scanner (Affymetrix).
|
| Data processing |
The scanned images were analysed using software resident in GeneChip Operating System v2.0 (Affymetrix). Sample loading and variations in staining were standardized by scaling the average of the fluorescent intensities of all genes on an array to a constant target intensity (250). The signal intensity for each gene was calculated as the average intensity difference, represented by [Σ(PM − MM)/(number of probe pairs)], where PM and MM denote perfect-match and mismatch probes.
The scaled gene expression values from GeneChip Operating System v2.0 software were imported into GeneSpring 7.2 software (Agilent Technologies) for preprocessing and data analysis. Probe sets were deleted from subsequent analysis if they were called absent by the Affymetrix criterion and displayed an absolute value below 20 in all experiments. The expression value of each gene was normalized to the median expression of all genes in each chip as well as the median expression for that gene across all chips in the study. Pairwise comparison of gene expression was performed for each matched experiment. Among direct comparisons between matched clinical isolates, genes were considered to be differentially expressed if their change in expression was ≥ 1.5-fold in three independent experiments.
|
| |
|
| Submission date |
Mar 22, 2013 |
| Last update date |
Jul 02, 2013 |
| Contact name |
Katherine S Barker |
| E-mail(s) |
ksbarker@uthsc.edu
|
| Organization name |
University of Tennessee Health Science Center
|
| Department |
Clinical Pharmacy
|
| Lab |
David Rogers laboratory
|
| Street address |
881 Madison Avenue, Room 305
|
| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38163 |
| Country |
USA |
| |
|
| Platform ID |
GPL6808 |
| Series (1) |
| GSE45438 |
Mitochondrial two-component signaling systems in Candida albicans |
|
