|
| Status |
Public on Dec 12, 2012 |
| Title |
Reverse Trans Expt 2 - MBY38 + no Dox 0 hrs - repeat 3 - Rev. fluor. |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Reverse Transition Expt - Cy5 Reference Sample
|
| Organism |
Candida albicans |
| Characteristics |
strain: MBY38
genotype/variation: TetO-UME6
other: Reverse Transition Pooled Reference: Equal aliquots of the 22 sample RNAs (Dox dosage of 20 ug/ml (n=11) or 0 ug/ml (n=11) at time points from 0 to 10 hours) 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 |
Reverse Transition Expt - MBY38 + no Dox 0 hr - Cy3
|
| Organism |
Candida albicans |
| Characteristics |
strain: MBY38
genotype/variation: TetO-UME6
|
| Treatment protocol |
Treatment time: 0 hours
Treatment temperature: 30 C
In-vitro treatment: In order to carry out the reverse hyphal-pseudohyphal-yeast transition experiment, the tetO-UME6 strain was grown overnight in 1.5 L YEPD medium at 30°C in the absence of Dox. When the culture reached OD600 ~ 0.01, Dox was added to a concentration of 20 µg/mL and cells were harvested for RNA extraction at each hour for 10 hours. Cells for the 0 hour time point were collected immediately prior to the addition of Dox. Over the time course cells were also harvested from an additional control culture in which the tetO-UME6 strain was grown in the same manner but in the absence of Dox.
|
| 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 |
GenePix Scanning Protocol
Other: Arrays were scanned using an Axon GenePix 4000B (Serial No.: 82713) microarray scanner along with GenePix Pro 6.0.1.25 image analysis software (Molecular Devices, Sunnyvale, CA).
|
| Description |
No additional information.
|
| Data processing |
Reverse Transition 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 reverse hyphal-pseudohyphal-yeast transition time course experiment was repeated twice (2 independent biological replicates). For each biological replicate, first separate median signal ratios (ratio of the median signal intensity of each spot) for +Dox and -Dox zero time point versus reference were determined based on 3 technical replicates each (one of which was performed using reverse fluors) using cDNA from the same total RNA preparation. Next, signal ratios for each time point sample versus reference were divided by the signal ratio for the corresponding zero time point versus reference.
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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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