|
| Status |
Public on Sep 11, 2007 |
| Title |
0.2 nM alpha factor 5min |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S.cerevisiae a-type cells 5 minutes after addition of alpha factor (0.2nM)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: W303-1a, bar1delta, haploid
|
| Growth protocol |
Cells were grown on YPD to log phase at 30oC prior to addition of alpha-factor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
5 O.D. units of cells were collected, pelleted and immediately frozen. Total RNA was obtained using RNeasy yeast RNA purification kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
The cDNA from alpha-factor treated and untreated cells was synthesized from total RNA using M-MLV Reverse Transcriptase RNase H Minus (Promega) and labeled with Cy3 and Cy5, respectively by the indirect amino-allyl method.
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| |
|
| Channel 2 |
| Source name |
S. cerevisiae grown in YPD at 30oC
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
For reference we used the same culture prior to addition of alpha-factor, in which cells were grown to log phase in YPD at 30oC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
5 O.D. units of cells were collected, pelleted and immediately frozen. Total RNA was obtained using RNeasy yeast RNA purification kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase RNase H Minus (Promega) and labeled with Cy3 and Cy5, respectively by the indirect amino-allyl method.
|
| |
|
| |
| Hybridization protocol |
Arrays were purchased from the Microarray Centre, University Health Network, Ontario, where PCR products are printed. For each hybridization, cDNA samples were labeled with Cy3 and Cy5 and combined with blockers: 5?g Herring sperm (Promega), 5?g tRNA (Gibco) and 17.5?g Poly A (Poly A oligos were synthesized with mixed length of 40, 50 and 60 Adenine residues). The labeled cDNAs were concentrated to 40?l using Microcon (Millipore) and 40?l of hybridization x2 solution (10x SSC, 50% formamide and 0.2% SDS) was added. Microarrays containing all yeast ORFs were pre-hybridized by incubating at 42ºC for 45 minutes in a solution containing 1% BSA, 25% formamide, 5x SSC and 0.1% SDS. The slides were washed in sterile water and dried by centrifugation (3 minutes, 2000 rpm). The labeled samples were boiled for 5 minutes, centrifuged for 1 minute, hybridized on the slide and placed in a hybridization chamber (Corning) for overnight incubation at 42ºC. The slides were then washed for 5 minutes at 42ºC with a solution containing 2x SSC and 0.1% SDS. Additional wash was performed at room temperature with a solution containing 0.1x SSC and 0.1% SDS, followed by three additional washes at room temperature in 0.1x SSC solution.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using ScanArray 4000 scanner (Packard BioScience) and image intensity data were extracted and analyzed with QuantArray analysis software.
|
| Description |
S.cerevisiae a-type cells were compared before and after (5 minutes) addition of alpha factor (0.2nM).
|
| Data processing |
For each array, bad spots were omitted from the analysis. For each spot, the Quantarray program outputs the level of signal intensity and background fluorescence. As a first step, we re-defined the spot intensity by subtracting the spot-specific background. For each channel (Cy3 or Cy5) we then calculated the value of background signal, defined by the mean (intensity-background) of the controlled spots printed with 3x SSC buffer. All intensity values below this background level were reset to this background level. The data for each spot was then transformed into a log2-ratios. The data was normalized by subtracting from each log2ratio the median. Values of duplicates were averaged.
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| |
|
| Submission date |
Sep 07, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Ofer Sarig |
| E-mail(s) |
[email protected]
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Lab |
Barkai
|
| Street address |
Weizmann Institute of Science
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL5823 |
| Series (1) |
| GSE8982 |
Mating response - six alpha factor concentrations (0.06, 0.2, 0.6, 6, 60 and 600 nM) |
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