|
| Status |
Public on Mar 15, 2008 |
| Title |
Resistant Isolate C56, Experiment 3 |
| Sample type |
RNA |
| |
|
| Source name |
Candida albicans isolate C56, mid-log phase
|
| Organism |
Candida albicans |
| Characteristics |
Fluconazole resistant isolate C56
|
| Biomaterial provider |
Dominique Sanglard
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot Phenol Method
|
| Label |
biotin
|
| Label protocol |
cRNA was synthesized and labelled 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 Labelling 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-labelled ribonucleotides, dithtiothreitol, RNase inhibitor mix and T7 RNA polymerase for 5 h at 37°C. The labelled 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. albicans NimbleExpress 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) and the fluorescence intensities were determined using the GCS 3000 high-resolution confocal laser scanner (Affymetrix).
|
| Scan protocol |
The scanned images were analysed using software resident in GeneChip Operating System v2.0 (GCOS; 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).
|
| Description |
Each resistant isolate was compared to its susceptible parent, and the TAC1 knockout was compared to the susceptible parent (5457) of the resistant isolate, 5674.
|
| Data processing |
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.
|
| |
|
| Submission date |
Aug 08, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Katherine S Barker |
| E-mail(s) |
[email protected]
|
| 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 |
GPL5723 |
| Series (1) |
| GSE8727 |
Genome-wide expression and location analyses of the Candida albicans Tac1p regulon |
|
