|
| Status |
Public on Dec 31, 2011 |
| Title |
WT4(Cy3) vs. WT_Blap 4 (Cy5) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wt BY4741 strain in log phase culture
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: by4741
genotype: : MATa, his3Δ1, leu2Δ0, LYS2, met15Δ0, ura3Δ0
|
| Biomaterial provider |
Mauricio Menacho-Marquez
|
| Treatment protocol |
Cells were grown at 28ºC in YPD rich medium until Ab660=0.6. 50 ml of yeast culture was treated with vehicle (1% DMSO) for 1 hour. Cells were then harvested by centrifugation and washed with cold water. Dried cell pellet was kept at-80 until RNA purification
|
| Extracted molecule |
total RNA |
| Extraction protocol |
standard protocol (acid-phenol extraction).
|
| Label |
Cy3
|
| Label protocol |
Indirect labelling
|
| |
|
| Channel 2 |
| Source name |
Wt BY4741 strain in log phase culture treated with b-lapachone
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: by4741
genotype: : MATa, his3Δ1, leu2Δ0, LYS2, met15Δ0, ura3Δ0
|
| Biomaterial provider |
Mauricio Menacho-Marquez
|
| Treatment protocol |
Cells were grown at 28ºC in YPD rich medium until Ab660=0.6. 50 ml of yeast culture was treated with 10 ug/ml b-lapachone in DMSO for 1 hour. Cells were then harvested by centrifugation and washed with cold water. Dried cell pellet was kept at-80 until RNA purification
|
| Extracted molecule |
total RNA |
| Extraction protocol |
standard protocol (acid-phenol extraction).
|
| Label |
Cy5
|
| Label protocol |
Indirect labelling
|
| |
|
| |
| Hybridization protocol |
Slides were hybridised using manual chambers according to manufacturer. Briefly, slides were prehybridized in 5 x SSC,0.1% SDS, and 0.1 mg/mL BSA for 45m, and rinse in 0.1 x SSC two times before drying.
Both samples were resuspended in 50% formamide, 5 x SSC,
0.1% SDS, and 0.1 mg/mL of sonicated salmon sperm DNA, denatured for 5 m and injected between slide and coverslip (Lifterslips). Hyb was performed overnight at 42ºC.
Washes
a) Arrays were inmersed in 2 x SSC, 0.1% SDS at 42°C until the
coverslip moves freely away from the slide.
b) Transferred to 2 x SSC, 0.1% SDS at 42°C for 5 minutes.
c) Transferred to 0.1 x SSC, 0.1% SDS at room temperature
for 5 minutes. Repeat step
d) Transferred to 0.1 x SSC at room temperature for
1 minute. Repeated 4 times
e) Rinsed in 0.01 x SSC for 10 seconds.
9. Dried by centrifugation at 1,600 x g for 2 minutes.
|
| Scan protocol |
Slides were scanning at 10um resolution using a 4200B Molecular Devices scanner, adjusting PMT values so that ratio Cy5/Cy3 total intensity is close to 1.
Image analysis was performed using GenePix 6.0 software.
|
| Description |
no further description
|
| Data processing |
ocal background was used for background sustraction. Background-substrated signals were used for ratio calculations. Data were normalized by global-median and print-tip loess. Features whose background-substrated intensity do not reach two times background intensity were flagged as bad and rejected for further analysis (Value=null). Equal for features affected by hyb and washing artefacts, if any. Original value data are log ratios of lowess-normalized Cy5/Cy3 signals.
|
| |
|
| Submission date |
Mar 26, 2009 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Jose Gadea |
| E-mail(s) |
jgadeav@ibmcp.upv.es
|
| Organization name |
IBMCP
|
| Department |
Stress
|
| Street address |
Ingeniero Fausto Elio
|
| City |
Valencia |
| ZIP/Postal code |
46022 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4069 |
| Series (1) |
| GSE15412 |
Yeast response to the DNA damaging beta-lapachone |
|
