|
| Status |
Public on Feb 27, 2015 |
| Title |
∆zta1_vs_wt_H2O2_repl2_dye_swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
wild type yeast, 30 minute treatment with 10mM hydrogen peroxide
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: FY834α
treatment: treated 30 min with 10 mM hydrogen peroxide
|
| Treatment protocol |
Cultures were treated with or without 10 mM hydrogen peroxide treatment for 30 min.
|
| Growth protocol |
Yeast cells from wild-type FY834α and ∆zta1 strains were grown to exponential phase (OD600≈0.7-0.8) in rich YPD medium at 30ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with RiboPure™-Yeast Kit from Ambion, following the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
Five-hundred nanograms of total RNA from each sample were reverse transcribed after priming with Oligo-dT- and labelled by in vitro transcription with T7 RNA polymerase in the presence of Cy5-CTP or Cy3-CTP using the Quick Amp Labeling Kit (Agilent) and purified using RNAeasy columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
mutant yeast, defective for ZTA1 (YBR046C) zeta-crystallin, 30 minute treatment with 10mM hydrogen peroxide
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: ∆zta1
treatment: treated 30 min with 10 mM hydrogen peroxide
|
| Treatment protocol |
Cultures were treated with or without 10 mM hydrogen peroxide treatment for 30 min.
|
| Growth protocol |
Yeast cells from wild-type FY834α and ∆zta1 strains were grown to exponential phase (OD600≈0.7-0.8) in rich YPD medium at 30ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with RiboPure™-Yeast Kit from Ambion, following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Five-hundred nanograms of total RNA from each sample were reverse transcribed after priming with Oligo-dT- and labelled by in vitro transcription with T7 RNA polymerase in the presence of Cy5-CTP or Cy3-CTP using the Quick Amp Labeling Kit (Agilent) and purified using RNAeasy columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
After fragmentation, 825 ng of cRNA labelled with two different dyes were co-hybridized on the same array using in situ hybridization buffer (Agilent) for 17 h, at 65ºC, and washed at room temperature for 1 min in Gene Expression Wash Buffer 1 (Agilent), and 1 min at 37ºC in Gene Expression Wash Buffer 2 (Agilent).
|
| Scan protocol |
Fluorescent images were obtained using an Agilent G2565BA scanner at 100% photomultiplier tube intensity and 100% laser power settings.
|
| Description |
∆zta1 vs wt, H2O2 stress, biological replicate 2 of 3, technical replicate 2 of 2
|
| Data processing |
TIFF image spot intensities were extracted and quantified with GenePix 6.0 software (Axon, Molecular Devices, Sunnyvale, CA) using the irregular-feature-finding option. The extracted raw data were processed with Bioconductor project software developed in the R statistical programming environment [Gentleman et al., 2004] using the Limma package [Smyth and Speed, 2003]. After normexp background subtraction was applied [Ritchie et al., 2007], the two channels on each array were balanced by lowess normalization using 0.3 as the span parameter with reduced weights in control and poor-quality spots, followed by a scaling step to make all arrays comparable,. For statistical assessment of differential gene expression, the Statistical Analysis of Microarrays (SAM) algorithm was used by applying the samr library in R [Tusher et al., 2001]. All analysis was performed using a Web interface implementation of the R-based software by Lozano and collaborators (unpublished data). Log2 ratio values from data obtained upon microarray analysis for all replicate probes of the same gene were reduced to a single value corresponding to the median of the log2ratios of all spots. Preliminary statistical analysis comparing the log2ratios from the six arrays from untreated samples with those of the 6 arrays from treated samples revealed no significant differences associated to hydrogen peroxide treatment likely due to the low dose used. Since there was no discernible effect by hydrogen peroxide, we were able to focus on the Zta1 dependent changes in gene expression that could help us understand the function of this gene. We pooled all 12 array datasets in order to increase the statistical power to detect any differences between both strains.
|
| |
|
| Submission date |
Mar 19, 2013 |
| Last update date |
Feb 28, 2015 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9294 |
| Series (1) |
| GSE45297 |
Yeast zeta-crystallin: An AU-Rich Element Binding Protein Involved in the Nutritional Stress Response |
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