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Sample GSM701081 Query DataSets for GSM701081
Status Public on May 23, 2011
Title Ssa_H5Rip_20d_replicate 3
Sample type RNA
 
Source name Ssa_H5Rip_20d
Organism Salmo salar
Characteristics tissue: Head-kidney
time: 20d
Extracted molecule total RNA
Extraction protocol Total RNA from RNAlater preserved salmon head-kidney samples was extracted using TRI reagent (Molecular Research Center, OH, USA) followed by column purification using the NucleoSpin RNA II (Macherey-Nagel, PA, USA) and following the manufacturer’s protocol. An on column DNase digestion step was performed on each sample. RNA quantification was performed using a NanoDrop 8000 (Thermo Scientific, DE, USA) and RNA quality and integrity was assessed with the Experion automated electrophoresis system (Bio-Rad, TX, USA). Only RNA samples with RNA Quality Indicator (RQI) values above 8 were used for analysis. RNA’s were stored at -70ºC in DEPC treated water containing an RNase inhibitor (Qiagen, ON, Canada) until needed.
Label Cy3
Label protocol RNA amplification and labeling for microarray was performed using the one-color Low Input Quick Amp Labeling kit (Agilent, CA, USA) and following the manufacturer’s recommendations. Briefly, 200 ng of each RNA sample was reverse transcribed, amplified and labeled using Cy3 dyes resulting in Cy3 labeled amplified RNA (aRNA). Purification of the labeled aRNA was performed using NucleoSpin RNA clean up columns (Macherey-Nagel, CA, USA) following the manufacturer’s recommendations. Following purification, labeled aRNA samples were then quantified using the NanoDrop 8000 to verify the yield of aRNA and the labeling efficiency. 1.65 µg of each purified aRNA was prepared for hybridization using the fragmentation step described in Agilent’s Low Input Quick Amp labeling kit protocol. Briefly each aRNA was fragmented by mixing 1.65 µg of aRNA in a volume of 22.8 µl, with 6 µl of 10X blocking agent and 1.2 µl of 25X fragmentation buffer and heating the mix at 60°C for 30 min.
 
Hybridization protocol Microarray hybridizations were performed using Agilent’s 4 x 44K Atlantic salmon microarray slides containing 4 grids with the same ~ 44 000 features (Design ID 020938), using a one-color approach. All hybridizations, including washes and drying steps were carried out on a HS4800 automated hybridization station using quad chambers (Tecan, Männedorf, Switzerland). Briefly, slides were initially washed with Agilent’s aOligo aCGH Prehybridization buffer for 1 min at 65°C and injected with a 60 µl of hybridization mix containing 30 µl of fragmentation mix and 30 µl of Agilent’s 2x GEx hybridization Buffer Hi-RPM per chamber. The injected material was hybridized for 17 hour at 65°C. Following hybridization slides were washed twice with Agilent’s gene expression wash buffer 1 at 23°C for 1 min, twice with Agilent’s gene expression wash buffer 2 with 0.01 wash buffer additive at 37°C for 1 min and then dried under nitrogen
Scan protocol Microarray slides were scanned at a resolution of 5 µm using a ScanArray Gx microarray scanner (Perkin Elmer, MA, USA) with the laser power set at 90%. For each slide, PMT settings were set to 60.
Description 252093810041_3_r10-44
Data processing TIFF images were quantified using the SpotReader feature extraction software (Niles Scientific, CA, USA). Numerical intensities from each spot were extracted and descriptive statistics were calculated and exported in a GenePix Results (GPR) file. Features that did not meet certain default criteria in SpotReader were flagged as bad. These default criteria included flagging spots which had more than 50 % of the pixels saturated, spots with less than 8 pixels in diameter and spots with high background The bad flag values computed were then used to filter out genes that had missing values (-50,-75,-100 GPR flag values) on more than half of the slides used in each group (e.g. H5Rc fish) for a particular statistical analysis performed. Median intensity value minus background for each spot remaining after filtering were log2 transformed and normalized using a median centering approach.
 
Submission date Apr 04, 2011
Last update date May 23, 2011
Contact name Francis LeBlanc
E-mail(s) [email protected]
Phone (506) 851-6222
Organization name Fisheries & Oceans Canada
Department Aquatic Animal Health
Lab Molecular Biology Unit
Street address 343 University Ave.
City Moncton
State/province NB
ZIP/Postal code E1C 9B6
Country Canada
 
Platform ID GPL7303
Series (1)
GSE28357 Transcriptional response of Atlantic salmon (Salmo salar) after primary versus secondary exposure to Infectious Salmon Anemia Virus (ISAV)

Data table header descriptions
ID_REF
VALUE Log 2 normalised median values -background

Data table
ID_REF VALUE
A_05_P246154 11.96825981
A_05_P246334 13.12097359
A_05_P247994 6.631752968
A_05_P248684 11.82096004
A_05_P248689 9.453526497
A_05_P248819 8.328720093
A_05_P248824 9.640181541
A_05_P248829 14.34658909
A_05_P248834
A_05_P248839
A_05_P248844
A_05_P248851 9.089134216
A_05_P248854 7.971603394
A_05_P248859
A_05_P248864
A_05_P248869
A_05_P248874 7.750694275
A_05_P248879 8.139547348
A_05_P248884
A_05_P248889 8.242777824

Total number of rows: 43663

Table truncated, full table size 830 Kbytes.




Supplementary file Size Download File type/resource
GSM701081.gpr.gz 2.0 Mb (ftp)(http) GPR
Processed data included within Sample table

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