|
| Status |
Public on Dec 22, 2006 |
| Title |
Candida glabrata transcription profile when grown in human urine (Exp 1-2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. glabrata grown in synthetic complete medium
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
Log phase C. glabrata cells grown in synthetic complete medium (SC medium, see Qbiogene catalog)
|
| Growth protocol |
C. glabrata wild type Bg2 cells were inoculate into 200 ml SC medium with an initial O.D.600 of 0.1. The cells were harvested after an incubation at 30C in a floor shaker (150 rpm) for 4 hrs when O.D. reached 0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell pellet was disrupted using 0.5 mm glass beads in a fast bead-beater. Total RNA was extracted using acid phenol. The RNA was further purified by RNeasy Mini kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
20 ug of total RNA was used to synthesize Cy5-cDNA probes with an anchor primer [(dT)18(dA/dG/dC)] in the presence of Cy5-dCTP (PerkinElmer) using SuperscriptTM III reverse transcriptase (Invitrogen). The probe was purified using MinElute TM reaction Cleanup kit (Qiagen) and vaccum dried.
|
| |
|
| Channel 2 |
| Source name |
C. glabrata grown in urine sample #1 (Exp 1-2)
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
Log phase C. glabrata cells grown in human urine supplemented with 2% glucose
|
| Growth protocol |
C. glabrata wild type Bg2 cells were inoculate into 200 ml human urine sample #1 (supplemented with 2% glucose and filter sterilized) with an initial O.D.600 of 0.1. The cells were harvested after incubation at 30C in a floor shaker (150 rpm) for 5.5 hrs when O.D. reached 0.55.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
same as above
|
| Label |
Cy3
|
| Label protocol |
same as above
|
| |
|
| |
| Hybridization protocol |
Both Cy5- and Cy3- cDNA probes were resuspened into hybridization buffer (35% formamide, 5*SSC, 0.1%SDS, 0.1 mg/ml denatured salmon sperm DNA) and combined to a total volume of 40 ul. The probe mix was denatured at 100 C for 10 s and quickly cooled on ice, then it was added on a microarray slides. The slides was covered with a cover glass and incubated in a chamber (Corning) at 42 C for 16-18 hrs. The washing protocol follows the manual for UltraGAPS coated slides (Corning).
|
| Scan protocol |
Hybridized slides was scaned by Axon 4000B microarray scanner with 100% power and PMT ranging 600-800. Data were acquired by Axon GenePix 5.0 software.
|
| Description |
This is experiment 2 of a dye-swap experiment. Experiment 1 is deposited in GSM151934.
|
| Data processing |
The median signal captured from Cy5-channel (F635) and from Cy3-channel (F532) was used to calculate C. glabrata gene expression change when grown in human urine.
|
| |
|
| Submission date |
Dec 20, 2006 |
| Last update date |
Dec 21, 2006 |
| Contact name |
Brendan Cormack |
| E-mail(s) |
bcormack@jhmi.edu
|
| Phone |
410-955-3651
|
| Organization name |
Johns Hopkins Univ. School of Medicine
|
| Department |
Molecular Biology & Genetics
|
| Street address |
Hunterian Building 617, 725 N. Wolfe St.
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL3922 |
| Series (1) |
| GSE6582 |
Candida glabrata transcription profile when grown under Niacin starvation or in human urine |
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