|
| Status |
Public on Jul 18, 2017 |
| Title |
t0_fluc_beg_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
candida albicans, t0, beginning, replicate2
|
| Organism |
Candida albicans |
| Characteristics |
strain: T0
treatment: not treated
time: beginning
|
| Treatment protocol |
Resistance to antifungal drugs was monitored using the E-test assay method (AB biodisk, Biomerieux). Therefore, cells were grown to mid-log phase, washed in 1xPBS, and diluted to an OD600 of 0.015. 150μl of cells were plated on SD minus uracil agar plates (pH7) using glass beads and allowed to dry for 15-30 minutes before applying a fluconazole E-test strip (0.016– 256 μg/ml, AB Biodisk). Plates were incubated at 30°C and the MIC was determined after 48 hours. Each passage the Fluconazole (Sigma Aldrich) concentration of the liquid (SD minus uracil) growth medium was adjusted to a concentration corresponding to twice the last measured MIC. Passages were pursued until strains were growing in liquid media containing a Fluconazole concentration of 256 μg/ml.
|
| Growth protocol |
Candida albicans strains used in this study were engineered to exhibit different levels of Leucine misincorporation at Serine CUG codons. T0: control strain 3,2% Leu misincorporation (natural mistranslation); T1: 24,9% Leu misincorporation. These highly ambiguous strains were constructed on the basis of the strain SN148 (arg4 /arg4 leu2 /leu2 his1 /his1 ura3 ::imm43/ura3 ::imm43 iro1 ::imm43/iro1 ::imm43) using homologous recombination methodologies as described in Bezerra et al PNAS 2013.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from strains at the beginning (T0, T1), middle (T0FM, T1FM) and end (T0FH, T1FH) of the evolution experiment. Additionally,clones were evolved for an equal number of total passages without the drug to study effects caused by mistranslation and by the antifungal fluconazole (T0NF, T1NF). RNA was extracted from mid-exponential phase cells using the hot phenol method. DNase I (Invitrogen) treated RNA was resuspended in RNAse free water and RNA quantity and integrity was assessed using UV/Vis spectrometry (Nanodrop) and the 2100 Agilent Bioanalyzer respectively.
|
| Label |
Cy3
|
| Label protocol |
Same amounts of RNA extract (500 ng) from each replicate were amplified and Cy-3-labeled using the Low Input Quick Amp Labeling kit (Agilent Technologies)
|
| |
|
| Hybridization protocol |
Custom gene expression SurePrint G3 microarrays (eArray accession number Candida_albicans_2007_1244213353206) were hybridized with CY3 labeled cRNA of growth duplicates according to manufacturer’s instructions (Agilent, Low Input Quick Amp Labelling kit).
|
| Scan protocol |
Arrays were scanned on an Agilent G2505C microarray scanner and microarray scan data was extracted using Agilent feature extraction software.
|
| Description |
t0Ub
|
| Data processing |
Gene expression microarray raw data were normalized using limmaGUI software (R/ Bioconductor, Boston, MA, USA).
|
| |
|
| Submission date |
Aug 05, 2014 |
| Last update date |
Jul 18, 2017 |
| Contact name |
Tobias Weil |
| E-mail(s) |
tobias.weil@fmach.it
|
| Organization name |
Fondazione Edmund Mach
|
| Street address |
Via E. Mach, 1
|
| City |
S. Michele all'Adige (TN) |
| ZIP/Postal code |
38010 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19049 |
| Series (2) |
| GSE60121 |
Mistranslation accelerates the evolution of antifungal drug resistance in Candida albicans [Gene expression] |
| GSE60122 |
Mistranslation accelerates the evolution of antifungal drug resistance in Candida albicans |
|
