|
| Status |
Public on Mar 31, 2006 |
| Title |
S. kudriavzevii Heat shock (37oC) 60 minutes |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
S. kudriavzevii Heat shock (37oC) 60 minutes
|
| Organism |
Saccharomyces kudriavzevii |
| Characteristics |
Strain: IFO 1802 wild type, diploid
|
| Growth protocol |
Cells were grown on YPD to log phase at 24oC prior to heat shock and then transferred to 37oC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
5 O.D. units of cells were collected, pelleted and immediately frozen. Total RNA was obtained using MasterPure yeast RNA purification kit (Epicenter).
|
| Label |
Cy5
|
| Label protocol |
The cDNA from heat shock 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.
|
| |
|
| Channel 2 |
| Source name |
S. kudriavzevii grown in YPD at 24oC
|
| Organism |
Saccharomyces kudriavzevii |
| Characteristics |
For reference we used the same culture prior to pertubation in which cells were grown to log phase in YPD at 30oC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA (20 ?g) was annealed with 4 ?g oligo dT12-18, and reverse-transcribed into cDNA with M-MLV Reverse Transcriptase RNase H Minus (Promega) for 2h at 45?C in the presence of 2.5mM MgCl2, 0.5 mM of dATP, dCTP and dGTP, 0.1mM dTTP, 0.4mM amino-allyl dUTP. Additional 400U M-MLV Reverse Transcriptase RNase H Minus was added and incubated for another 2 hours. After hydrolysis of RNA in 0.2 M NaOH (30min, 65oC), Tris was removed from the reaction with a Microcon-30 concentrator.
|
| 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 |
none provided
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using Aginlet's DNA microarray scanner and image intensity data were extracted and analyzed with SpotReader (Niles Scientific) analysis software.
|
| Description |
S. kudriavzevii Heat shock (37oC) 60 minutes
|
| Data processing |
Background intensity was subtracted using a Bayesian correction, and the ratios of the two dyes were log2-transformed. Log2 ratios were then corrected for intensity-dependant and spatial-dependant biases by subtracting a Lowess curve followed by a median filter. The two spots corresponding to each gene were then averaged, and genes for which the two spots were significantly different were declared as 'missing values' (along with other genes which were flagged by the image analysis software or removed by manual inspection).
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| |
|
| Submission date |
Oct 03, 2005 |
| Last update date |
Feb 07, 2008 |
| Contact name |
Naama Barkai |
| E-mail(s) |
naama.barkai@weizmann.ac.il
|
| Organization name |
The Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Lab |
Naama Barkai
|
| Street address |
Hertzel
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL2910 |
| Series (1) |
| GSE3406 |
Expression patterns in stress conditions of 4 closely related yeast species from the Saccharomyces sensu stricto complex |
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