|
| Status |
Public on Mar 24, 2009 |
| Title |
Wild-type Cells 27 hours following mtDNA loss compared to Wild-type cells with mtDNA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT cells with intact mtDNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: UCC1899
|
| Growth protocol |
Cells were grown in YEPD medium at or below an OD of 1.0 for the duration of the experiment
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using acid phenol
|
| Label |
Cy5, Cy3
|
| Label protocol |
labeled cDNA targets were prepared by reverse transcription of 2 µg of mRNA using oligo(dT)18 primer in the presence of 0.2 mM 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (Sigma-Aldrich Company, St. Louis, Mo.), 0.3 mM dTTP, and 0.5 mM each dATP, dCTP, and dGTP. Following cDNA synthesis, either Cy3 or Cy5 monoreactive fluor (Amersham Life Sciences, Arlington Heights, Ill.) was covalently coupled to the cDNA-incorporated aminoallyl linker in the presence of 50 mM sodium bicarbonate (pH 9.0).
|
| |
|
| Channel 2 |
| Source name |
WT cells 27 hours following loss of mtDNA by treatment with 30micrograms/ml ethidum bromide for 6 hours
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: UCC1899
|
| Growth protocol |
Cells were grown in YEPD medium at or below an OD of 1.0 for the duration of the experiment
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using acid phenol
|
| Label |
Cy3, Cy5
|
| Label protocol |
labeled cDNA targets were prepared by reverse transcription of 2 µg of mRNA using oligo(dT)18 primer in the presence of 0.2 mM 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (Sigma-Aldrich Company, St. Louis, Mo.), 0.3 mM dTTP, and 0.5 mM each dATP, dCTP, and dGTP. Following cDNA synthesis, either Cy3 or Cy5 monoreactive fluor (Amersham Life Sciences, Arlington Heights, Ill.) was covalently coupled to the cDNA-incorporated aminoallyl linker in the presence of 50 mM sodium bicarbonate (pH 9.0).
|
| |
|
| |
| Hybridization protocol |
Hybridization was carried out for 16 hours at 63 degrees celsius in 0.2% SDS/3X SSC. Slides were then washed with 1x SSC/0.03% SDS, then 1x SSC, then 0.2x SSC, then 0.05x SSC and then dried.
|
| Scan protocol |
Following cohybridization to the chip, a fluorescent image of a microarray was collected at both emission wavelengths using a GenePix 4000 fluorescence scanner (Axon Instruments, Inc., Foster City, Calif.), and image analysis was performed using GenePix Pro microarray acquisition and analysis software
|
| Description |
average over 6 samples, 3 biological replicates per dye swap
|
| Data processing |
LOWESS normalized using agilent software (Genespring 7)
|
| |
|
| Submission date |
Mar 19, 2009 |
| Last update date |
Mar 23, 2009 |
| Contact name |
Daniel Gottschling |
| E-mail(s) |
[email protected]
|
| Phone |
206-667-4494
|
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Department |
Division of Basic Sciences
|
| Street address |
1100 Fairview Avenue N., Mailstop A3-025
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL1914 |
| Series (1) |
| GSE15302 |
Cells following mtDNA loss, or NAR1 repression |
|
