|
| Status |
Public on Jun 22, 2009 |
| Title |
CJD21/PMK-CAP1 + Benomyl (expt 3) |
| Sample type |
RNA |
| |
|
| Source name |
CAP1-deficient strain with CAP1 reintroduced, benomyl-treated
|
| Organism |
Candida albicans |
| Characteristics |
strain: CJD21/PMK-CAP1 (wildtype CAP1 allele)
experimental treatment: benomyl, 25 mcg/ml, 30min
|
| Biomaterial provider |
Martine Raymond
|
| Treatment protocol |
Cultures were grown to saturation overnight in YPD at 30 deg Celsius with agitation. Optical density was adjusted to 0.2, and cells were grown as before in YPD for 3hrs. Benomyl was then added to a final concentration of 25 mcg/ml, and cells were incubated an additional 30min prior to harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected by centrifugation. Cell pellets were frozen at -80 deg Celsius for at least 30min, and RNA was isolated from the 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 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-labeled ribonucleotides, dithtiothreitol, RNase inhibitor mix, and T7 RNA polymerase for 5 h at 37°C. The labeled cRNA was separated from unincorporated ribonucleotides with 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 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 C. albicans NimbleExpress GeneChip arrays (Affymetrix). 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). The scanned images were analyzed 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 of 250.
|
| Description |
Array is first-generation custom C. albicans Affymetrix chip manufactured by NimbleGen.
|
| 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, respectively.
|
| |
|
| Submission date |
Dec 16, 2008 |
| Last update date |
Jun 04, 2010 |
| 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) |
| GSE14258 |
Identification of the Candida albicans CAP1 regulon |
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