|
| Status |
Public on Mar 14, 2008 |
| Title |
tetO-TBF1/tbf1 vs TBF1/tbf1 profiling 5h-tetracycline replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
tetO-TBF1/tbf1 treated with tetracycline (100ug/ml) for 5h
|
| Organism |
Candida albicans |
| Characteristics |
Strain: tetO-TBF1/tbf1
|
| Treatment protocol |
Tetracycline was added at 100ug/ml for 5h.
|
| Growth protocol |
YPD medium to OD600 of 1.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by the hot phenol extraction method (Carlson and Botstein, 1982) with the difference that glass beads (Sigma) were added to samples. Total RNA was subsequently cleaned up on RNeasy columns (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of Cy5-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript II reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
TBF1/tbf1 Heterozygous strain treated with tetracycline (100ug/ml) for 5h
|
| Organism |
Candida albicans |
| Characteristics |
Strain: TBF1/tbf1
|
| Treatment protocol |
Tetracycline was added at 100ug/ml for 5h.
|
| Growth protocol |
YPD medium to OD600 of 1.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by the hot phenol extraction method (Carlson and Botstein, 1982) with the difference that glass beads (Sigma) were added to samples. Total RNA was subsequently cleaned up on RNeasy columns (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of Cy3-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript II reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
The microarray slides were pre-hybridized for 2 hours at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. The slides were washed twice with SSC-0.2% SDS, once with 0.1X SSC-0.2% SDS and 3 times with 0.1X SSC. A final wash with isopropanol was performed and slides were air-dried.
|
| Scan protocol |
Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 10-µm resolution.
|
| Description |
Arrays are organized in 48 sub-arrays containing 280 spots each.
|
| Data processing |
Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).
|
| |
|
| Submission date |
Feb 12, 2008 |
| Last update date |
Feb 23, 2008 |
| Contact name |
Hugo Lavoie |
| E-mail(s) |
hugo.lavoie@nrc-cnrc.gc.ca
|
| Phone |
514-496-6154
|
| Fax |
514-496-6213
|
| Organization name |
CNRC-IRB
|
| Department |
Genetics
|
| Street address |
6100 Royalmount
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H4P 2R2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6475 |
| Series (2) |
| GSE10499 |
Transcription Factor Substitution during the Evolution of Fungal Ribosome Regulation_expression profiling |
| GSE10622 |
Transcription Factor Substitution during the Evolution of Fungal Ribosome Regulation |
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