|
| Status |
Public on Dec 12, 2012 |
| Title |
Control Expt - PCY87 + 20 ug/ml Dox - repeat 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Dosage Expt - Cy3 Reference Sample
|
| Organism |
Candida albicans |
| Characteristics |
strain: MBY38
genotype/variation: TetO-UME6
other: Dosage Expt Pooled Reference Protocol: Equal aliquots of the 12 sample RNAs (Dox dosage: 20 ug/mL, 1 ug/mL, 0.4 ug/mL, 0.35 ug/mL, 0.3 ug/mL, 0.25 ug/mL, 0.2 ug/mL, 0.15 ug/mL, 0.1 ug/mL, 0.05 ug/mL, 0.02 ug/mL, 0 ug/mL ) were combined to create a pooled reference sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot Acid Phenol Extraction Protocol
Other: RNA was isolated from C. albicans cells using the hot acid phenol method. Total RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturer's recommendations.
|
| Label |
cy3
|
| Label protocol |
Cy3 Labeling Protocol
Other: Secondary hybridization was carried out using the complimentary capture reagents provided in the 3DNA Array 350 kit (Genisphere). For each reaction, the following were added: 3DNA capture reagent with Cy3 (2.5 uL), 3DNA capture reagent with Cy5 (2.5 uL), SDS-based hybridization buffer (vial 6-Genisphere) (26 uL), and RNase/DNnase-free water (21 uL). The secondary hybridization solution was incubated in the dark at 80 C for 10 min., then at 50 C for 15 min.
|
| |
|
| Channel 2 |
| Source name |
Control Expt - PCY87 + 20 ug/ml Dox - Cy5
|
| Organism |
Candida albicans |
| Characteristics |
strain: PCY87
genotype/variation: tetR-HAP4
|
| Treatment protocol |
Treatment type: compound
Agent: Doxycycline (Dox)
Treatment dose: 20.0 ug/mL
Treatment time: Overnight
Treatment temperature: 30 C
In-vitro treatment: In order to determine the effect of Dox alone on C. albicans global gene expression, the PCY87 control strain was grown to saturation and diluted into 50 mL YEPD medium in the presence or absence of 20 µg/mL Dox. These cultures were grown overnight at 30°C and cells were harvested for RNA preparation at OD600 = 1.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot Acid Phenol Extraction Protocol
Other: RNA was isolated from C. albicans cells using the hot acid phenol method. Total RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies) according to manufacturer's recommendations.
|
| Label |
cy5
|
| Label protocol |
Cy5 Labeling Protocol
Other: Secondary hybridization was carried out using the complimentary capture reagents provided in the 3DNA Array 350 kit (Genisphere). For each reaction, the following were added: 3DNA capture reagent with Cy3 (2.5 uL), 3DNA capture reagent with Cy5 (2.5 uL), SDS-based hybridization buffer (vial 6-Genisphere) (26 uL), and RNase/DNnase-free water (21 uL). The secondary hybridization solution was incubated in the dark at 80 C for 10 min., then at 50 C for 15 min.
|
| |
|
| |
| Hybridization protocol |
Sample Hybridization Protocol
Other: Hybridization was performed by adding 48 uL of secondary hybridization solution to the slide under a supported glass coverslip and carried out at 65 C for 3 hr. at high humidity in the dark. At hybridization termination, arrays were gently submerged into 2X SSC, 0.2% SDS (at 65 C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. To prevent fluorophore degradation, the arrays were treated with Dyesaver (Genisphere).
|
| Scan protocol |
GenePix and ScanArray Scanning Protocol
Other: Arrays were scanned using an Axon GenePix 4000B (Serial No.: 82713) microarray scanner along with ScanArray Express 2.00 image analysis software.
|
| Description |
No additional information.
|
| Data processing |
Dox Control Expt Data Processing
Calculation Method: Microarray data was normalized using the Lowess method (Y.H. Yang, 2001) and filtered using Partek software to only include spots showing a median signal intensity >200 and signal-to-noise ratio >2. For the experiment to determine the effect of Dox alone on C. albicans global gene expression, signal ratios for the minus Dox sample versus reference were divided by signal ratios for the 20 ug/mL Dox sample versus reference. This experiment was performed using 5 independent biological replicates. Three of the biological replicates were labeled using normal fluors, one of which was repeated as a technical replicate using reverse fluors. Two of the 5 biological replicates were performed using reverse fluors.
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| |
|
| Submission date |
Jul 26, 2012 |
| Last update date |
Dec 12, 2012 |
| Contact name |
David Kadosh |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Texas Health Science Center at San Antonio
|
| Department |
Microbiology and Immunology
|
| Lab |
Kadosh Lab 5.023V
|
| Street address |
7703 Floyd Curl Drive
|
| City |
San Antonio |
| State/province |
TX |
| ZIP/Postal code |
78229 |
| Country |
USA |
| |
|
| Platform ID |
GPL15843 |
| Series (1) |
| GSE39677 |
A Genome-wide Transcriptional Analysis of Morphology Determination in Candida albicans |
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