|
| Status |
Public on Sep 28, 2006 |
| Title |
Pfu gamma 20min replicate3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pfu irradiated
|
| Organism |
Pyrococcus furiosus |
| Characteristics |
P. furiosus (DSMZ 3638) cells gamma irradiated
|
| Biomaterial provider |
Williams
|
| Treatment protocol |
Cultures grown to approximately 5x106 cells/ml were chilled to 4 C and exposed to 2,500 Gy of gamma radiation using a 26,000-curie (9.6x1014 Bq) 60Co gamma source at the University of Maryland College Park Gamma Test Facility, at a dose rate of 73 Gy/min. Cultures were further incubated at 90 C and samples for RNA extraction were taken 20 min after the temperature of the cultures reached 90 C (DiRuggiero et al. 1997).
|
| Growth protocol |
Cells were grown in 100ml serum bottles at 90 C under anaerobic conditions in the absence of sulfur and with 100 M Na2WO4 and 0.5% (wt/vol) maltose
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were centrifuged at 4000xg for 4 min at 4 C and RNA was extracted from the pellet using the TRI reagent (SIGMA T9424, St Louis, MO) as previously described (Chomczynski and Sacchi 1987). RNA samples were analyzed with a Beckman DU640 spectrophotometer (Beckman Coulter, Fullerton, CA) and gel electrophoresis for quality and quantification.
|
| Label |
Cy5
|
| Label protocol |
First strand cDNA synthesis and labeling was carried out with total RNA (3 g), 200U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), random hexamers, 0.5 mM dATP, dGTP, dCTP, 0.2mM dTTP and 0.3 mM Cy3-dUTP or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ) for 110 min at 42 C. Labeled cDNAs were cleaned up with alkaline treatment and Cyscribe GFX columns (Amersham Biosciences) according to the manufacturer s instructions.
|
| |
|
| Channel 2 |
| Source name |
Pfu reference
|
| Organism |
Pyrococcus furiosus |
| Characteristics |
P. furiosus (DSMZ 3638) cells non-irradiated
|
| Biomaterial provider |
Williams
|
| Treatment protocol |
Cultures were grown to approximately 5x106 cells/ml and RNA was immediately extracted.
|
| Growth protocol |
Cells were grown in 100ml serum bottles at 90 C under anaerobic conditions in the absence of sulfur and with 100 M Na2WO4 and 0.5% (wt/vol) maltose
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were centrifuged at 4000xg for 4 min at 4 C and RNA was extracted from the pellet using the TRI reagent (SIGMA T9424, St Louis, MO) as previously described (Chomczynski and Sacchi 1987). RNA samples were analyzed with a Beckman DU640 spectrophotometer (Beckman Coulter, Fullerton, CA) and gel electrophoresis for quality and quantification.
|
| Label |
Cy3
|
| Label protocol |
First strand cDNA synthesis and labeling was carried out with total RNA (3 g), 200U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), random hexamers, 0.5 mM dATP, dGTP, dCTP, 0.2mM dTTP and 0.3 mM Cy3-dUTP or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ) for 110 min at 42 C. Labeled cDNAs were cleaned up with alkaline treatment and Cyscribe GFX columns (Amersham Biosciences) according to the manufacturer s instructions.
|
| |
|
| |
| Hybridization protocol |
Hybridization was carried out in a sealed Corning hybridization chamber (Acton, MA) at 60 C for 18 hours. For each experimental time point four slides, with at least two features per open reading frame (ORF), were hybridized producing 8 replicates or more per ORF.
|
| Scan protocol |
Slides were scanned using an Axon 4100a Genepix personal slide scanner and the Genepix software V5 (Axon, Union City, CA).
|
| Description |
For each time point, 2 types of replicates were included: (i) technical replicates: each gene-specific PCR product was spotted at 2 spatially different locations onto the slides, (ii) biological replicates: each comparison was performed from 4 independently processed cultures and hybridized to 4 different slides (replicate 1 to 4) to account for inherent biological variation occurring independently of the experimental perturbations, giving a total of 8 data points per condition per gene.
|
| Data processing |
The background-normalized intensities were normalized by median ratio normalization as follows: median values were calculated for the ratios of the intensities for the two channels, channel one was divided by the square root of the median and channel two was multiplied by the square root of the median. Ratio of fluorescence intensity between sample and reference RNA were calculated as log10 values
|
| |
|
| Submission date |
Aug 17, 2006 |
| Last update date |
Sep 27, 2006 |
| Contact name |
Jocelyne DiRuggiero |
| E-mail(s) |
diruggie@umd.edu
|
| Phone |
301-405-4598
|
| Fax |
301-314-9081
|
| Organization name |
University of Maryland
|
| Department |
Cell Biology and Molecular Genetics
|
| Street address |
3221 H.J. Patterson Hall
|
| City |
College Park |
| State/province |
MD |
| ZIP/Postal code |
20742 |
| Country |
USA |
| |
|
| Platform ID |
GPL3926 |
| Series (1) |
| GSE5919 |
Microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus exposed to gamma irradiation |
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