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Sample GSM455439 Query DataSets for GSM455439
Status Public on Mar 01, 2010
Title HKC1R
Sample type RNA
 
Channel 1
Source name head kidney, control fish
Organism Sparus aurata
Characteristics tissue: head kidney
fish: 1
weight: 185g
length: 18.75cm
tank: 21-2
pathogen: Enteromyxum leei
pathogen exposure: control
Biomaterial provider Jaume Pérez-Sánchez, Ariadna Sitjà-Bobadilla, Josep Calduch: GinerInstituto de Acuicultura de Torre de la Sal (CSIC) 12595 Ribera de Cabanes, Castellón, Spain
Treatment protocol One fish group was exposed to E. leei-contaminated effluent (recipient group = R). R fish (n= 66, average weight = 134 g) were placed in two replicated 200L fibre-glass tanks which were set to receive exclusively the effluent water from another tank containing 24 infected (donors = D; average weight = 127.3 g; prevalence of infection = 54%) gilthead sea bream. The D to R fish ratio was 0.8. Other 66 naïve fish were allocated in two replicated tanks (control group = CTRL) under the same conditions, but without receiving contaminated effluent. Over the course of the study, day length followed natural changes and water was heated in order to keep temperature always above 18 oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight. Disease signs and daily mortalities were recorded throughout the experiments. The parasitic status of dead fish was checked by microscopic examination of fresh intestinal scrapings. Fish were sampled after 113 days post exposure. Feeding was stopped one day prior to the sampling to ensure that the digestive tract was empty. Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80 oC until RNA extraction and analysis.
Growth protocol Gilthead sea bream were checked for the absence of the parasite and acclimated to the experimental conditions two weeks before the beginning of the experiment. Day length followed natural changes and water was heated in order to keep temperature always above 18oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight.
Extracted molecule total RNA
Extraction protocol Qiagen RNeasy Maxi combination protocol with Qiazol reagent
Label Cy3
Label protocol Ten micrograms of total RNA per individual fish (control/experimental) and ten micrograms of reference RNA per slide were indirectly labelled following an "in house" protocol with the red fluorescent dye ester cyanine 5 (Cy5) and green fluorescent dye ester Cyanine 3 (Cy3) both from GE Healthcare (UK). In brief, cDNA was synthesised using Superscript III MMLV reverse transcriptase (Invitrogen) incorporating aminoallyl dUTP (Sigma Aldrich). Synthesis took place at 42 oC for 2 hours. RNA was hydrolysed with 10M NaOH/0.5M EDTA for 15 minutes and the reaction was then neutralised with 1M HEPES. Reverse transcription reactions were purified using Microcon 30 columns (Millipore, UK) according to manufacturer~s instructions. Purified cDNAs were dried using a vacuum concentrator (Eppendorf) and were then resuspended in 0.1M sodium bicarbonate buffer. Cy dyes were resuspended in the same buffer and coupling to the Cy dye ester took place in the dark for 2 hours at room temperature. The removal of unincorporated dye was performed using the Illustra CyScribe GFX purification kit (GE Healthcare). Labelled cDNA samples were tested for successful dye incorporation using a Nanodrop spectrophotometer (Nanodrop technologies).
 
Channel 2
Source name reference pool of all RNA (except hki1, hki2, hki3, hkc7, hkn2, hkn3, hkn4, hkn5, hkn7, intn1, intn5, intn8, hkc7, hkc8, hki1, hki2, hki3, hki6, intc3, intc5, intc6)
Organism Sparus aurata
Characteristics tissue: head kidney
sample type: reference
Biomaterial provider Jaume Pérez-Sánchez, Ariadna Sitjà-Bobadilla, Josep Calduch: GinerInstituto de Acuicultura de Torre de la Sal (CSIC) 12595 Ribera de Cabanes, Castellón, Spain
Treatment protocol One fish group was exposed to E. leei-contaminated effluent (recipient group = R). R fish (n= 66, average weight = 134 g) were placed in two replicated 200L fibre-glass tanks which were set to receive exclusively the effluent water from another tank containing 24 infected (donors = D; average weight = 127.3 g; prevalence of infection = 54%) gilthead sea bream. The D to R fish ratio was 0.8. Other 66 naïve fish were allocated in two replicated tanks (control group = CTRL) under the same conditions, but without receiving contaminated effluent. Over the course of the study, day length followed natural changes and water was heated in order to keep temperature always above 18 oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight. Disease signs and daily mortalities were recorded throughout the experiments. The parasitic status of dead fish was checked by microscopic examination of fresh intestinal scrapings. Fish were sampled after 113 days post exposure. Feeding was stopped one day prior to the sampling to ensure that the digestive tract was empty. Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80 oC until RNA extraction and analysis.
Growth protocol Gilthead sea bream were checked for the absence of the parasite and acclimated to the experimental conditions two weeks before the beginning of the experiment. Day length followed natural changes and water was heated in order to keep temperature always above 18oC, the range was 18-23 oC. Water was 5 micrometer-filtered and UV irradiated, and salinity was 37.5~. Water flow was 10L/min and oxygen content of outlet water remained higher than 85%saturation. All fish were fed daily a commercial dry pellet diet at about 1% of body weight.
Extracted molecule total RNA
Extraction protocol Qiagen RNeasy Maxi combination protocol with Qiazol reagent
Label Cy5
Label protocol Ten micrograms of total RNA per individual fish (control/experimental) and ten micrograms of reference RNA per slide were indirectly labelled following an "in house" protocol with the red fluorescent dye ester cyanine 5 (Cy5) and green fluorescent dye ester Cyanine 3 (Cy3) both from GE Healthcare (UK). In brief, cDNA was synthesised using Superscript III MMLV reverse transcriptase (Invitrogen) incorporating aminoallyl dUTP (Sigma Aldrich). Synthesis took place at 42 oC for 2 hours. RNA was hydrolysed with 10M NaOH/0.5M EDTA for 15 minutes and the reaction was then neutralised with 1M HEPES. Reverse transcription reactions were purified using Microcon 30 columns (Millipore, UK) according to manufacturer~s instructions. Purified cDNAs were dried using a vacuum concentrator (Eppendorf) and were then resuspended in 0.1M sodium bicarbonate buffer. Cy dyes were resuspended in the same buffer and coupling to the Cy dye ester took place in the dark for 2 hours at room temperature. The removal of unincorporated dye was performed using the Illustra CyScribe GFX purification kit (GE Healthcare). Labelled cDNA samples were tested for successful dye incorporation using a Nanodrop spectrophotometer (Nanodrop technologies).
 
 
Hybridization protocol The two cDNA samples per slide (e.g. reference cDNA Cy3 labelled and experimental cDNA Cy5 labelled) were mixed and appropriate blockers were added (sheared salmon sperm genomic DNA, poly dA). The cDNA mixture was concentrated by precipitation with 3M sodium acetate and absolute ethanol at -20oC for 30 min. followed by centrifugation at 12500 x g for 30 min. Following removal of supernatant, the cDNA pellet was resuspended in 70 µl slide hyb buffer #1 (Ambion) and was then denatured at 95oC for 2 min. Slides were placed in Pre-Hyb solution (3xSSC, 0.1 % BSA, 0.1 % SDS) in 50 ml Sarsted tubes pre-warmed @ 42 °C and pre-hybed @ 42 °C for 30 min. Slides were rinsed 3 times in water and spun dry for 3 min @ 1600rpm. The hybridisation mixture was then added to the pre-hybed microarray slide using a coverslip (Erie Scientific). Hybridisation was performed in a Genetix hybidisation chamber (Genetix, UK) at 42 °C for 16 h. Following hybridisation the slides were washed (2 x 5 min. washes using 0.2XSSC/0.1% SDS and 2 x 5 min. washes using 0.2XSSC) using an Advawash automatic washing station (Advalytix).
Scan protocol Microarray slides were scanned in two channels (543 & 633nm) at 5 microM resolution using a confocal laser scanner (ScanArray Express, Perkin Elmer)
Description Head kidney and posterior intestine were rapidly excised, frozen in liquid nitrogen, and stored at -80oC until RNA extraction and analysis.
Data processing TIFF image files were imported into Genepix Pro (version 5.0.1.24) (Axon instruments) for feature finding and alignment using a batch alignment process. Features were flagged as present, absent or bad by this software program and pixel intensities for feature and background were quantified. Output Genepix results (GPR) files were imported into the Genespring GX 7.3 software program (Agilent technologies). 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 using 20.0% of the data 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.
 
Submission date Sep 23, 2009
Last update date Mar 04, 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 GPL8467
Series (2)
GSE18219 Aquafirst_seabream_pathogen exposure_head kidney
GSE20619 Gilthead seabream pathogen exposure to Enteromyxum leei

Data table header descriptions
ID_REF
VALUE log2 [(Cy3 Median signal - Cy3 Median background) / (Cy5 Median signal - Cy5 Median background)]
CH1_MEDIAN channel 1 signal intensity median - Cy3
CH1_MEAN channel 1 signal intensity mean
CH1_SD channel 1 signal intensity standard deviation
CH1_BKD_MEDIAN channel 1 background signal intensity median
CH1_BKD_MEAN channel 1 background signal intensity mean
CH1_BKD_SD channel 1 background signal intensity standard deviation
CH2_MEDIAN channel 2 signal intensity median - Cy5
CH2_MEAN channel 2 signal intensity mean
CH2_SD channel 2 signal intensity standard deviation
CH2_BKD_MEDIAN channel 2 background signal intensity median
CH2_BKD_MEAN channel 2 background signal intensity mean
CH2_BKD_SD channel 2 background signal intensity standard deviation
FLAGS
PRE_VALUE (Cy5 Median signal - Cy5 Median background) / (Cy3 Median signal - Cy3 Median background), averaged for the duplicate spots
INV_VALUE log2 [(Cy5 Median signal - Cy5 Median background) / (Cy3 Median signal - Cy3 Median background)]

Data table
ID_REF VALUE CH1_MEDIAN CH1_MEAN CH1_SD CH1_BKD_MEDIAN CH1_BKD_MEAN CH1_BKD_SD CH2_MEDIAN CH2_MEAN CH2_SD CH2_BKD_MEDIAN CH2_BKD_MEAN CH2_BKD_SD FLAGS PRE_VALUE INV_VALUE
af_sb_Guide Dot 200ng_microliter 1.0058 803 841 314 138 170 96 345 411 314 148 218 171 0 0.498 -1.0058
af_sb_Guide Dot 200ng_microliter_1 1.0058 875 889 334 146 192 342 338 403 305 146 223 230 0 0.498 -1.0058
af_sb_Guide Dot 200ng_microliter_2 1.0058 1111 1140 415 138 185 335 356 453 326 146 233 245 0 0.498 -1.0058
af_sb_3xSSC+1,5MBetaine 0.1125 172 203 118 138 176 120 172 251 203 153 232 208 -100 0.925 -0.1125
af_sb_Guide Dot 100ng_microliter 0.6326 898 881 321 135 162 85 379 441 292 136 210 180 0 0.645 -0.6326
af_sb_Guide Dot 50ng_microliter 0.5929 975 982 352 136 161 81 441 484 321 134 204 204 0 0.663 -0.5929
af_sb_Guide Dot 5ng_microliter 0.4442 313 345 198 129 156 86 234 312 243 134 211 205 0 0.735 -0.4442
af_sb_cdn09p0005/O12/cdn09p0005o12.f.1 0.6171 592 625 294 126 154 85 332 390 276 130 201 159 0 0.652 -0.6171
af_sb_cdn10p0006/K4/cdn10p0006k04.f.1 -0.2314 344 376 173 133 163 86 296 375 277 151 229 185 0 1.174 0.2314
af_sb_cdn12p0001/N1/cdn12p0001n01.f.1 -0.6772 394 425 201 156 189 110 420 505 325 214 293 226 0 1.599 0.6772
af_sb_cdn13p0004/K10/cdn13p0004k10.f.1 -0.2228 3903 3756 1171 147 181 111 2173 2216 972 183 266 221 0 1.167 0.2228
af_sb_cdn14p0005/J23/cdn14p0005j23.f.1 -0.2869 244 286 171 158 217 181 227 292 211 154 234 199 -100 1.22 0.2869
af_sb_gsgl01b15/G6/gsgl01b15g06r1_m13rev.1 1.0029 822 855 339 155 220 179 372 426 283 155 232 183 0 0.499 -1.0029
af_sb_gsgy07b22/I19/gsgy07b22i19r1_m13rev.1 -0.0854 9076 8374 2643 137 189 178 4385 4276 1605 147 234 375 0 1.061 0.0854
af_sb_cdn01p0002/H20/cdn01p0002h20.f.1 -0.338 688 732 299 132 173 166 492 573 366 150 237 407 0 1.264 0.3380
af_sb_cdn02p0005/P3/cdn02p0005p03.f.1 0.1877 549 565 234 136 162 83 344 461 396 145 220 181 0 0.878 -0.1877
af_sb_cdn03p0006/C1/cdn03p0006c01.f.1 -0.4468 688 739 297 140 166 84 528 616 351 141 213 169 0 1.363 0.4468
af_sb_cdn04p0006/M9/cdn04p0006m09.f.1 -0.2016 1582 1560 593 144 172 88 956 1021 544 149 220 172 0 1.15 0.2016
af_sb_cdn06p0003/A4/cdn06p0003a04.f.1 null 261 285 155 141 168 85 224 277 195 143 216 173 -100 null null
af_sb_cdn07p0004/A24/cdn07p0004a24.f.1 0.2584 643 674 255 146 175 92 383 457 308 154 226 178 0 0.836 -0.2584

Total number of rows: 38976

Table truncated, full table size 4374 Kbytes.




Supplementary file Size Download File type/resource
GSM455439.gpr.gz 4.3 Mb (ftp)(http) GPR
Processed data included within Sample table

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