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Sample GSM125332 Query DataSets for GSM125332
Status Public on Sep 28, 2006
Title Pfu gamma 20min replicate1
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 100µM 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 (3µg), 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 100µM 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 (3µg), 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 07, 2006
Last update date Sep 27, 2006
Contact name Jocelyne DiRuggiero
E-mail(s) [email protected]
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

Data table header descriptions
ID_REF
VALUE normalized log10 values
CH1_SIG_MEDIAN median values for channel 1 fluorescence intensities at 635 nm
CH1_BKD_MEDIAN median values for channel 1 fluorescence intensities at 635 nm for background
CH2_SIG_MEDIAN median values for channel 2 fluorescence intensities at 532 nm
CH2_BKD_MEDIAN median values for channel 2 fluorescence intensities at 532 nm for background

Data table
ID_REF VALUE CH1_SIG_MEDIAN CH1_BKD_MEDIAN CH2_SIG_MEDIAN CH2_BKD_MEDIAN
ID-1 -0.181385669 277 154 247 76
ID-2 -0.30590091 235 154 225 75
ID-3 -0.624560394 198 149 257 68
ID-4 -0.138122686 235 146 180 68
ID-5 -0.267631441 207 138 183 66
ID-6 -0.230499883 243 146 219 68
ID-7 -0.315190986 186 149 137 67
ID-8 -0.411206673 273 148 364 69
ID-9 -0.594173675 203 146 273 68
ID-10 -0.200684651 307 146 298 64
ID-11 -0.469170736 216 150 244 66
ID-12 -0.089094824 287 150 220 66
ID-13 -0.17710981 311 157 279 67
ID-14 -0.162162396 352 149 336 66
ID-15 0.048208271 1793 154 1410 67
ID-16 -0.426438147 1226 150 2699 69
ID-17 -0.520379344 234 147 331 67
ID-18 -0.498321627 244 149 340 66
ID-19 -0.515728479 614 151 1459 69
ID-20 -0.067258366 448 158 382 72

Total number of rows: 9216

Table truncated, full table size 287 Kbytes.




Supplementary data files not provided

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