|
| Status |
Public on Nov 04, 2011 |
| Title |
C_albicans_WT_v_bcr1_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from Candida albicans BCR1 knockout cells, grown in SD medium supplemented with 50 mM glucose and 10% feotal calf serum and labeled with Cyanine-5
|
| Organism |
Candida albicans |
| Characteristics |
age: 5h
growth condition: 37 degree Celsius, SD medium supplemented with 50 mM glucose and 10% feotal calf serum
strain: CJN702
genotype/variation: BCR1 knockout
|
| Growth protocol |
Overnight cultures of C. albicans CJN702 were diluted to a A600 of 0.2 in 50 ml of SD medium supplemented with 50 mM glucose and 10% feotal calf serum, and culture was incubated at 200 rpm at 37 degree for 5 hours
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion)
|
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA was used for cDNA labeling. Briefly RNA samples were incubated with 0.2 pmol of Candida albicans specific RT primers mix (Eurogentec) and 200 pmol of anchor oligonucleotide deoxyriboslthymine (Invitrogen) at 70°C for 10 min, after which following reaction reagents were added: 1 X First strand buffer, 20 µmol dATP, dTTP, and dGTP, 2 µmol dCTP, 400 pmol dithiothreitol, 400 U Superscript II reverse transcriptase (Invitrogen). 2 nmol Cy5-dCTP or Cy3-dCTP was then added into the mixture in the dark. The mixture was left at 42°C for two hours followed by adding additional 400 U Superscript II reverse transcriptase, and reaction was continued for one more hour. 50 µg/ml-1 RNase A and 0.05 U RNase H were added and the reaction was incubated for 30 min at 37°C.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from Candida albicans DAY286 cells in SD medium supplemented with 50 mM glucose and 10% feotal calf serum and labeled with Cy3
|
| Organism |
Candida albicans |
| Characteristics |
age: 5h
growth condition: 37 degree Celsius, SD medium supplemented with 50 mM glucose and 10% feotal calf serum
strain: DAY286
genotype/variation: wild type
|
| Growth protocol |
Overnight cultures of C. albicans DAY286 were diluted to a A600 of 0.2 in 50 ml of SD medium supplemented with 50 mM glucose and 10% feotal calf serum, and culture was incubated at 200 rpm at 37 degree for 5 hours
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion)
|
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA was used for cDNA labeling. Briefly RNA samples were incubated with 0.2 pmol of Candida albicans specific RT primers mix (Eurogentec) and 200 pmol of anchor oligonucleotide deoxyriboslthymine (Invitrogen) at 70°C for 10 min, after which following reaction reagents were added: 1 X First strand buffer, 20 µmol dATP, dTTP, and dGTP, 2 µmol dCTP, 400 pmol dithiothreitol, 400 U Superscript II reverse transcriptase (Invitrogen). 2 nmol Cy5-dCTP or Cy3-dCTP was then added into the mixture in the dark. The mixture was left at 42°C for two hours followed by adding additional 400 U Superscript II reverse transcriptase, and reaction was continued for one more hour. 50 µg/ml-1 RNase A and 0.05 U RNase H were added and the reaction was incubated for 30 min at 37°C.
|
| |
|
| |
| Hybridization protocol |
Purified cDNA was denatured at 95 degrees Celsius for 2 min, and then chilled on ice for 10 sec. Two cDNA samples were mixed. The hybridization was carried out using Agilent Gene Expression Hybridization Kit (Agilent Technologies) according to the manufacturer's manual.
|
| Scan protocol |
Scanned with an Axon 4000B scanner at 10 µm resolution. Data was acquired using GenePix 5.0 software. The quality and concentration of the isolated RNA was analyzed using an Agilent 2100 Bioanalyzer.
|
| Data processing |
LOWESS normalized, no background correction using the LIMMA package from Bioconductor.
|
| |
|
| Submission date |
Nov 03, 2011 |
| Last update date |
Nov 04, 2011 |
| Contact name |
Geraldine Butler |
| E-mail(s) |
geraldine.butler@ucd.ie
|
| Organization name |
University Colege Dublin Conway Institute
|
| Department |
School of Biomolecular and Biomedical Science
|
| Lab |
Butler lab
|
| Street address |
Belfield
|
| City |
Dublin |
| ZIP/Postal code |
D4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL10903 |
| Series (2) |
| GSE33460 |
Transcriptional profile of Candida albicans bcr1 knockout. |
| GSE33490 |
Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans |
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