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Sample GSM624404 Query DataSets for GSM624404
Status Public on Nov 24, 2010
Title LS45F
Sample type RNA
 
Channel 1
Source name LS45
Organism Oncorhynchus mykiss
Characteristics stress response: Low
fish number: 45
Biomaterial provider Dr Thomas G Pottinger, Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, United Kingdom
Treatment protocol A time-course study using HR and LR fish from the F3 generation was carried out during August and September 2004. During July, LR and HR fish (LR weight: 181.3 g; length: 24.7 cm; HR weight: 184.7 g; length: 24.9 cm) were distributed between twenty-two 1000 litre holding tanks (approx 110 fish/tank, 11 for each line). Each tank was supplied with a constant flow of untreated Windermere lake water (20 litres min-1) at ambient temperature (mean 16.7 oC). After a 2 week period of acclimation, LR fish from a single holding tank were transferred to twenty-two 50 litre confinement tanks, 5 fish per tank, each supplied with a constant flow of lake water. At intervals (0, 2, 4, 6, 8, 24, 48, 96, 168, 336, 504h) a total of 6 fish (3 fish from each of two tanks) were sampled, with undisturbed LR fish in pairs of holding tanks acting as controls. At each time-point livers were collected and immediately frozen in liquid nitrogen for subsequent analysis. The confinement and sampling procedure was repeated 1 week later for the HR fish. Fish were fasted for 2 days prior to the study and offered food on day 3 of the study and three times weekly thereafter. Plasma cortisol was measured by a validated radioimmunoassay procedure (Pottinger and Carrick, 2001).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using the RNeasy Midi Kit (Qiagen) following the manufacturer's recommended protocol after homogenising 0.2 - 0.3 mg of tissue in Qiazol reagent (Qiagen). Contaminating genomic DNA was degraded using an on-column DNase I treatment using RNase-free DNase Kit (Qiagen). The quality of RNA was checked using the Agilent Bioanalyser 2100. RNA integrity numbers (RIN) were 10 for most of the samples. Total RNA was quantified by UV spectrophotometry at 260 nm (Eppendorf Biophotometer).
Label Cy5
Label protocol Five microg of total RNA was used to synthesize labelled cDNA using the ChipShotTM Direct Labeling and Clean-Up System (Promega) following the manufacturer¢s protocol. Labelled cDNA was quantified using a NanoDropTM ND 1000 spectrophotometer (Thermo Scientific).
 
Channel 2
Source name REFERENCE
Organism Oncorhynchus mykiss
Characteristics reference: Pooled samples
Biomaterial provider Dr Thomas G Pottinger, Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, United Kingdom
Treatment protocol A time-course study using HR and LR fish from the F3 generation was carried out during August and September 2004. During July, LR and HR fish (LR weight: 181.3 g; length: 24.7 cm; HR weight: 184.7 g; length: 24.9 cm) were distributed between twenty-two 1000 litre holding tanks (approx 110 fish/tank, 11 for each line). Each tank was supplied with a constant flow of untreated Windermere lake water (20 litres min-1) at ambient temperature (mean 16.7 oC). After a 2 week period of acclimation, LR fish from a single holding tank were transferred to twenty-two 50 litre confinement tanks, 5 fish per tank, each supplied with a constant flow of lake water. At intervals (0, 2, 4, 6, 8, 24, 48, 96, 168, 336, 504h) a total of 6 fish (3 fish from each of two tanks) were sampled, with undisturbed LR fish in pairs of holding tanks acting as controls. At each time-point livers were collected and immediately frozen in liquid nitrogen for subsequent analysis. The confinement and sampling procedure was repeated 1 week later for the HR fish. Fish were fasted for 2 days prior to the study and offered food on day 3 of the study and three times weekly thereafter. Plasma cortisol was measured by a validated radioimmunoassay procedure (Pottinger and Carrick, 2001).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using the RNeasy Midi Kit (Qiagen) following the manufacturer's recommended protocol after homogenising 0.2 - 0.3 mg of tissue in Qiazol reagent (Qiagen). Contaminating genomic DNA was degraded using an on-column DNase I treatment using RNase-free DNase Kit (Qiagen). The quality of RNA was checked using the Agilent Bioanalyser 2100. RNA integrity numbers (RIN) were 10 for most of the samples. Total RNA was quantified by UV spectrophotometry at 260 nm (Eppendorf Biophotometer).
Label Cy3
Label protocol Five microg of total RNA was used to synthesize labelled cDNA using the ChipShotTM Direct Labeling and Clean-Up System (Promega) following the manufacturer¢s protocol. Labelled cDNA was quantified using a NanoDropTM ND 1000 spectrophotometer (Thermo Scientific).
 
 
Hybridization protocol Cy3-labelled and Cy5-labelled cDNA were dried and resuspended in 22 microL of hybridisation mix (5X Denhardt, 3.5X SSC, 0.3% SDS (Bio-Rad), 0.5 microg/microL yeast tRNA (Invitrogen), 0.5 microg/microL polyA RNA (Invitrogen) and 50 % formamide). Slides were prehybridized with a solution containing 50mM ethanolamine, 100mM Tris base, 0.1% SDS, pH 9.0 at 50 °C for 15 min followed by two washes in 18 MW water. Next slides were incubated in a solution containing 4X SSC and 0.1% SDS at 50 °C for 15 min followed by one wash with 18 MW water and centrifugation (260g) for 3 minutes at room temperature. Slides were incubated in prehybridisation solution (3.5X SSC, 0.3% SDS, 1% BSA) at 42 °C for 1h. Slides were washed 5 times with 18 MW water and dried by centrifugation (260g) for 3 min. Before hybridisation, Cy3- and Cy5-labelled targets were pooled and denatured at 100 °C for 2 min. Targets were applied to the microarray slide and covered with a coverslip (Erie Scientific). Hybridisation of the slides was performed in a hybridisation chamber (Genetix) humidified with 3X SSC at an annealing temperature of 42 °C overnight followed by immersion of slides in 2% SSC, 0.1% SDS for the removal of the coverslip. Slides were washed with 1X SSC for 2 min and followed by 2 washes with 0.2X SSC for 2 min each. Slides were dried by centrifugation (260g) for 3 min at room temperature and immediately scanned.
Scan protocol ScanArray Express HT Microarray Scanner (Perkin Elmer) - GenePix software (GenePix Pro 6.0.1.25) was used for feature acquisition from the TIF images (Molecular Devices Corp).
Description The fish used in the study were sexually immature, mixed sex, adult rainbow trout from two lines of fish selectively bred for either a high (HR) or low (LR) corticosteroid response to a standardized confinement stressor. The selection programme used to generate the HR and LR lines of trout has been described previously (Pottinger and Carrick, 1999 and 2001). In brief, single parent matings of individuals identified as either HR or LR by their corticosteroid response to a series of standardized confinement stressors were used to generate F1 lines of HR and LR progeny. An individual-within-family selection process was adopted to establish the F2 and subsequently the F3 generation, used in this study. The F3 generation hatched during spring 2003 and these fish were therefore in their second year (18 months old) at the time of this study. The mean weight and fork length of the two lines at the time of the study was LR: 181 g, 24.7 cm; HR: 185 g, 24.9 cm.
Data processing Data was processed in GeneSpring v7.3. Intensity values used were signal median values and local background was automatically subtracted. To account for dye swap, the signal channel and control channel measurements for appropriate samples were reversed. Values below 0.01 were set to 0.01. A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Genespring automatically averages spots with the same ID so all triplicate were averaged. Finally the output from GeneSpring is log2 transformed.
 
Submission date Nov 17, 2010
Last update date Nov 23, 2010
Contact name Michael Taylor Cairns
E-mail(s) michael.cairns@nuigalway.ie
Phone 0035391492094
Organization name NUI Galway
Department MRI
Lab Rm 104
Street address University Road
City Galway
ZIP/Postal code n/a
Country Ireland
 
Platform ID GPL11206
Series (2)
GSE25440 Individual variation in confinement response at 168h
GSE25584 Confinement stress in HR and LR trout lines

Data table header descriptions
ID_REF
VALUE log2 ratio test/reference

Data table
ID_REF VALUE
1 4.717
2 0.366
3 0.507
4 0.244
5 -1.165
6 0.544
7 -0.868
8 -5.305
9 -0.080
10 2.001
11 -0.297
12 -0.095
13 null
14 0.410
15 -0.336
16 3.614
17 1.048
18 -0.739
19 -2.102
20 -0.258

Total number of rows: 11088

Table truncated, full table size 124 Kbytes.




Supplementary file Size Download File type/resource
GSM624404.gpr.gz 1.3 Mb (ftp)(http) GPR
Processed data included within Sample table

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