|
| Status |
Public on Nov 11, 2010 |
| Title |
Control sample, replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Yeast cells exponentially grown in YPD medium and exposed to MOPS buffer
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: FY1679
treatment: no antimicrobial peptide
|
| Treatment protocol |
250 μl of a 100X stock solution of each peptide were added to each yeast culture (final concentration 5 μM). The same volume of MOPS buffer was added to the control sample. Cultures were grown at 30ºC with shaking for 3 additional hours. Three independent biological replicates were conducted for each treatment.
|
| Growth protocol |
25 ml cultures of 10E+05 colony-forming units (CFU)/ml of S. cerevisiae FY1679 were grown with shaking at 30ºC in 20% YPD medium (100% YPD is 1% yeast extract, 2% peptone and 2% dextrose) for three hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Yeast cells were collected by centrifugation and kept at 80ºC until processed for RNA isolation. Total RNA was extracted from cell pellets and ethanol precipitated.
|
| Label |
33P-dCTP
|
| Label protocol |
Radiolabelled cDNA was obtained by reverse transcription (RT) of 20 μg of total RNA, after annealing to 3.75 µg of the anchor oligonucleotide oligo(dT)VN (Invitrogen), in the presence of 5 mM DTT, 800 μM each of dATP, dTTP and dGTP, 5 μM dCTP, 5 μl of 3000 Ci/mmol α33P-dCTP, 10 units RNase inhibitor (Invitrogen), and 400 units SuperScript III reverse transcriptase (Invitrogen), at 50ºC for 2h. Template RNA was removed by alkaline hydrolysis, followed by neutralization. Unincorporated nucleotides were separated from the 33P-labelled cDNA probe by passage through MicroSpin S-300HR columns (Amersham).
|
| |
|
| Hybridization protocol |
The nylon filters from the macroarray containing 6,020 yeast ORFs (Laboratory of DNA chips, Universitat de València, http://scsie.uv.es/chipsdna/) were hybridized with 33P-labelled cDNA probes and stripped. A total of three different filters were used, and each biological replicate from each of the three treatments (control, 5 μM PAF26, and 5 μM melittin) was hybridized to a distinct filter. Therefore, each individual filter was subjected to three cycles of hybridization and stripping.
|
| Scan protocol |
Filters were exposed for 5–7 days to an imaging plate (BAS-MP 2040, FujiFilm), which was scanned in a phosphorimaging scanner (FLA-3000, FujiFilm).
|
| Description |
CTR_3
|
| Data processing |
Raw image quantitation background subtracted: Macroarray hybridization images were quantified with the software package ArrayVision v8.0 (Imaging Research Inc.). The local background was defined as the mean signal intensity of an area around each block of 16 hybridized spots, and subtracted from each signal.
Values were submitted to statistical analysis (ArrayStat v1.0 software) as described in López-García et al. (2010), BMC Microbiology.
|
| |
|
| Submission date |
Nov 10, 2010 |
| Last update date |
Nov 10, 2010 |
| Contact name |
MONICA GANDIA |
| E-mail(s) |
mgandia@iata.csic.es
|
| Organization name |
IATA
|
| Department |
CIENCIA DE LOS ALIMENTOS
|
| Lab |
FISIOLOGÍA Y BIOTECNOLOGÍA POSTCOSECHA
|
| Street address |
AVDA. AGUSTIN ESCARDINO 7
|
| City |
PATERNA |
| State/province |
VALENCIA |
| ZIP/Postal code |
46980 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4565 |
| Series (1) |
| GSE25279 |
A genomic approach highlights common and diverse effects and determinants of susceptibility on the yeast Saccharomyces cerevisiae exposed to distinct antimicrobial peptides |
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