|
| Status |
Public on Apr 18, 2011 |
| Title |
Candida_parapsilosis_control_vs_fluconazole_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from C. parapsilosis BC014S
|
| Organism |
Candida parapsilosis |
| Characteristics |
isolate: BC014S
growth condition: 35 degree celsius
medium: YPD medium
|
| Treatment protocol |
No treatment protocol.
|
| Growth protocol |
Overnight cultures of C. parapsilosis were diluted to a A600 of 0.2 in 50 ml of YPD medium and culture was incubated at 200 rpm at 35 degrees until A600 of 1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion). The quality and concentration of the isolated RNA was analyzed using an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Twenty-four micrograms of total RNA was used for cDNA labeling. Briefly, RNA samples were incubated with 100 pmol of anchor oligonucleotide deoxyriboslthymine (Invitrogen) at 70 degrees for 10 min, after which following reaction reagents were added including 1 X first strand buffer, 6.67 mM dATP, dTTP, and dGTP; 2mM dCTP; 100 mM dithiothreitol; 2 µl Superscript II reverse transcriptase (Invitrogen). 37.5 µM Cy3-dCTP were then added into the mixture in dark. The mixture were left at 42 degrees for two hours followed by adding additional 2 µl Superscript II reverse transcriptase, and reaction was continued for one more hour. 50 µg/ml RNase A and 0.05 unit/µl RNase H were added and the reaction was incubated for 30 min at 37 degrees.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from C. parapsilosis BC014FLC
|
| Organism |
Candida parapsilosis |
| Characteristics |
isolate: BC014FLC
growth condition: 35 degree celsius
medium: YPD medium
|
| Treatment protocol |
No treatment protocol.
|
| Growth protocol |
Overnight cultures of C. parapsilosis were diluted to a A600 of 0.2 in 50 ml of YPD medium and culture was incubated at 200 rpm at 35 degrees until A600 of 1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion). The quality and concentration of the isolated RNA was analyzed using an Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Twenty-four micrograms of total RNA was used for cDNA labeling. Briefly, RNA samples were incubated with 100 pmol of anchor oligonucleotide deoxyriboslthymine (Invitrogen) at 70 degrees for 10 min, after which following reaction reagents were added including 1 X first strand buffer, 6.67 mM dATP, dTTP, and dGTP; 2mM dCTP; 100 mM dithiothreitol; 2 µl Superscript II reverse transcriptase (Invitrogen). 37.5 µM Cy3-dCTP were then added into the mixture in dark. The mixture were left at 42 degrees for two hours followed by adding additional 2 µl Superscript II reverse transcriptase, and reaction was continued for one more hour. 50 µg/ml RNase A and 0.05 unit/µl RNase H were added and the reaction was incubated for 30 min at 37 degrees.
|
| |
|
| |
| Hybridization protocol |
Purified cDNA was denatured at 95 degrees 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 manufacture's manual.
|
| Scan protocol |
Scanned with an Axon 4000B scanner at 10 µm resolution. Data was acquired using GenePix 5.0 software.
|
| Data processing |
LOWESS normalized, no background correction using the LIMMA package from Bioconductor.
|
| |
|
| Submission date |
Feb 18, 2011 |
| Last update date |
Apr 18, 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 |
GPL13192 |
| Series (2) |
| GSE27405 |
Transcriptional response of an azole-resistant Candida parapsilosis isolate [fluconazole]. |
| GSE27409 |
Transcriptional response of an azole-resistant Candida parapsilosis isolate. |
|
