|
| Status |
Public on Dec 12, 2012 |
| Title |
Dosage Expt 1 - MBY38 + 20 ug/ml Dox - repeat 3 - Rev. fluor. |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Dosage Expt - Cy5 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 |
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.
|
| |
|
| Channel 2 |
| Source name |
Dosage Expt - MBY38 + 20 ug/ml Dox - Cy3
|
| Organism |
Candida albicans |
| Characteristics |
strain: MBY38
genotype/variation: TetO-UME6
|
| Treatment protocol |
Treatment type: compound
Agent: Doxycycline (Dox)
Treatment dose: 20 ug/ml
Treatment time: Overnight
Treatment temperature: 30 C
In-vitro treatment: In all experiments cells were grown under non-filament-inducing conditions: yeast extract-peptone-dextrose (YEPD) medium at 30°C. For the experiment in which C. albicans morphologies were examined in static culture in response to UME6 dosage, a saturated overnight culture of the tetO-UME6 (MBY38) strain was diluted into 50 mL of YEPD medium plus various concentrations of Doxycycline (Dox) (20, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.02 and 0 µg/mL) and grown overnight at 30°C to OD600 = 1.0. Cells were then harvested for RNA extraction.
|
| 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.
|
| |
|
| |
| 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 |
ScanArray Scanning Protocol
Other: Arrays were scanned using a ScanArray Express HT (Serial No.: 680078) microarray scanner along with ScanArray Express 2.00 image analysis software.
|
| Description |
No additional information.
|
| Data processing |
Dosage 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. The UME6 dosage experiment was performed using two independent biological replicates. In each biological replicate experiment, the median signal ratio (ratio of the median signal intensity of each spot) for the 20 ug/mL Dox sample versus reference sample was first determined based on three technical replicates (one of which was performed using reverse fluors) using cDNA prepared from the same total RNA preparation. Next, the signal ratio of each Dox concentration sample versus reference was divided by the median signal ratio of the 20 ug/mL Dox sample versus reference.
|
| |
|
| 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 |
|
