|
| Status |
Public on Dec 22, 2006 |
| Title |
Candida glabrata transcription profile in response to Niacin limitation (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 (containing 3.25 uM nicotinic acid)
|
| Growth protocol |
C. glabrata wild type Bg2 cells were inoculate into 250 ml synthetic complete medium (containing 3.25 uM of nicotinic acid) with an initial O.D.600 of 0.07. The cells were harvested at hr 5 after an incubation at 30C in a floor shaker (150 rpm) when O.D. reached 0.6.
|
| 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 under Niacin limitation
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
Log phase C. glabrata cells grown in synthetic medium containing 0.016 uM nicotinic acid
|
| Growth protocol |
C. glabrata wild type Bg2 cells were inoculate into 250 ml synthetic medium containing 0.016 uM of nicotinic acid with an initial O.D.600 of 0.06. The cells were harvested at hr 5.5 after an incubation at 30C in a floor shaker (150 rpm) when O.D. reached 0.65.
|
| 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 GSM151863.
|
| Data processing |
The median signal captured from Cy5-channel (F635) and from Cy3-channel (F532) was used to calculate C. glabrata gene expression change in response to Niacin limitation
|
| |
|
| Submission date |
Dec 19, 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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