|
| Status |
Public on Aug 28, 2011 |
| Title |
with 200 mg/L alachlor (IC20)_biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Cell population incubated with 200 mg/L alachlor (IC20)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
cell population: Exponential cells (O.D.640nm = 0.25 ± 0.05)
|
| Treatment protocol |
Cells were harvested by centrifugation (10000 rpm, 3 min, 0ºC) and immediately frozen in liquid nitrogen and stored at -80ºC until RNA extraction.
|
| Growth protocol |
Exponential cell populations (OD640nm = 0.25 ± 0.05) of S. cerevisiae BY4741 were incubated in minimal growth medium (pH 6.5), at 30ºC, with orgital agitation (250 RPM) in each of the exposure conditions indicated above.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was performed using the hot-phenol method (Koher & Domdey, 1991, Methods in Enzimology 194:398-405).
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Yeast Genome 2.0 Arrays, according to the manufacturer’s GeneChip 3’ IVT Express kit user manual. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls was used in a reverse transcription reaction (GeneChip 3’ IVT Express Kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in a 16h in vitro transcription (IVT) reaction to generate aRNA (GeneChip 3’ IVT Express Kit; Affymetrix). Size distribution of the aRNA and fragmented aRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
|
| |
|
| Hybridization protocol |
5 µg of fragmented aRNA was used in a 100-µl hybridization cocktail containing added hybridization controls. 80 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (FS450_0003; GeneChip HWS kit, Affymetrix) were used on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
Gene expression data from cells incubated with the IC20 of alachlor
|
| Data processing |
Scanned arrays were analyzed first with Affymetrix Expression Console software for quality control. Subsequent analysis was carried out with DNA-Chip Analyzer (dChip) 2010 (http://www.dchip.org, Wong Lab, Harvard) applying a probeset mask file excluding all probes on the array representing Schizosaccharomyces pombe transcripts. The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method (Li and Wong, 2001). Normalized CEL intensities of the 12 arrays were used to obtain model-based gene expression indices based on a PM (Perfect Match)-only model (Li and Hung Wong, 2001). Replicate data for the same sample type were weighted gene-wise by using inverse squared standard error as weights. All genes compared were considered to be differentially expressed if the 90% lower confidence bound of the fold change between experiment and baseline was above 1.2 .
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| |
|
| Submission date |
Apr 18, 2011 |
| Last update date |
Aug 28, 2011 |
| Contact name |
Cristina Anjinho Viegas |
| E-mail(s) |
[email protected]
|
| Phone |
+351218419180
|
| Fax |
+351218419199
|
| Organization name |
Instituto Superior Técnico
|
| Department |
Bioengineering
|
| Lab |
Biological Sciences
|
| Street address |
Av Rovisco Pais
|
| City |
Lisboa |
| ZIP/Postal code |
1049-001 Lisboa |
| Country |
Portugal |
| |
|
| Platform ID |
GPL2529 |
| Series (1) |
| GSE28677 |
Gene expression data from yeast exposure to alachlor |
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