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Sample GSM1289596 Query DataSets for GSM1289596
Status Public on Jul 31, 2016
Title Spleen_20ug/kg_rep5
Sample type RNA
 
Source name spleen, pool of 3
Organism Anguilla anguilla
Characteristics tissue: spleen
developmental stage: elver
diet: LPSp
dose: 20ug/kg
Treatment protocol Elvers (1.7 ± 0.2 g) obtained from local fishermen (Delta del Ebro, Spain) were fed at different doses of LPSp (0, 20 and 40 μg LPSp kg BW-1 day-1) for a period of 70 days (4 replicates per dietary group). LPSp was added to the diet (52% protein, 24% fat, 9% ash) by top coating the pellets with the lyophilized powder of LPSp dissolved in 2% fish oil (diets manufactured by ALLERQUA, Denmark). The trial was conducted in 100 L tanks (40 L functional volume) connected to a recirculation unit (IRTAmar®) and water quality was as follows: temperature, 21 ± 0.5 ºC; pH, 7.5 ± 0.1; photoperiod, 12 L:12 D and oxygen at saturation levels.
Extracted molecule total RNA
Extraction protocol Extraction method: TRIzol (Invitrogen)
Label Cy3
Label protocol One-Color Microarray-Based Gene Expression analysis (Low Input Quick Amp Labeling) Version 6.5 May 2010. Samples are retrotranscribed (first strand synthesis) and labeled using Low imput Quick Amp Labeling kit, One color (Agilent Technologies, Cat. Nº 5190-2305) following manufacturer instructions. First, total RNA is retrotranscribed with AffinityScript Reverse Transcriptase (AffinityScript RT), an engineered thermostable mutant derived from Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, using Oligo dT primers coupled to T7 promoter. Double stranded cDNA synthesized by AffinityScript RT is in vitro transcribed by T7 RNA pol in the presence of either Cy3-CTP or Cy5-CTP fluorophores to generate amplified and labeled cRNA. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen). Amount of nucleic acid labeled: 50 ng. After labeling and purification, cRNA is quantify with NanoDrop ND-1000 spectrophotometer in order to determine the yield and specific activity of each reaction. Yield: ug of cRNA. Should be > 0.825 ug. Specific activity: pmol Cy3 per ug of cRNA. Should be > 6.
 
Hybridization protocol Chamber type: SureHyb hybridization chamber (Agilent). Quantity of each labeled extract used: 600 ng. Volume: 40 uL. Temperature (ºC): 65. Duration: 17 hours at 10 rpm in the hybridization oven.
Scan protocol Scanned on a G2565CA DNA microarray scanner (Agilent Technologies), with ozone-barrier slide covers (Agilent P/N G2505-60550). Profiles AgilentG3_GX_1Color. SCAN C SETTINGS. Dye Channel:Green. Scan Region:61 x 21,6 mm. Scan resolution (um):3. Tiff:20 bit.
Description B5 (Dieta 20%)
Data processing Images (.tiff images) were quantified using Agilent Feature Extraction Software (ver. 10.7.3.1) (Agilent Technologies). Feature Extraction protocol for data extraction: GE1_107_Sep09. Design File/Grid Template: 035147_D_F_20110629. QC Metric Set: GE1_QCMT_Sep09.
Raw data from Feature Extraction software was subsequently processed on GeneSpring GX 12.6 (Agilent Technologies).That file contains the summarized data per biological feature and per sample. Preprocessing: First we transform all Feature Extraction flags to Absent/Marginal/Present calls (Compromised/Not Detected/Detected Flags in the new GeneSpring versions), the same way as we did with the Tissue Experiment: Feature is NOT Positive and significant: ABSENT (Compromised). Feature is NOT uniform: ABSENT (Compromised). Feature is NOT well above background: MARGINAL (Not Detected). Feature is Saturated: MARGINAL (Not Detected). Feature is Population outlier: ABSENT (Compromised). Threshold raw signals (lineal data) to 1. Summarization: calculate the geometric mean of replicated probes The resulted summarized lineal data is the “Raw Signal” in result files. Gene Spring uses the “gProcessedSignals” from Feature Extraction txt Raw Data Files).Log base2 transformation. Intra-chip normalization: quantile normalization. Baseline to Mean of Control (Dieta 0) Samples. Filter Probesets based on Present/Absent calls: Use flag spot information in data files. Entities retained in which 100 % of samples in 1 out of any 3 conditions (DIETA 0, DIETA 20%, DIETA 40%) have Present (Detected) or Marginal (Not Detected) Flag.
 
Submission date Dec 16, 2013
Last update date Jul 31, 2016
Contact name Ibon Cancio
E-mail(s) [email protected]
Organization name UPV/EHU
Street address B/sarriena s/n
City Leioa
ZIP/Postal code 48940
Country Spain
 
Platform ID GPL18071
Series (1)
GSE53346 Transcriptomic responses of Anguilla anguilla elvers fed dietary LPS

Data table header descriptions
ID_REF
VALUE quantile normalized signal intesities

Data table
ID_REF VALUE
CUST_4145_PI426546238 -0.21470165
CUST_7271_PI426544517 0.33972168
CUST_3322_PI426546238 -0.10964203
CUST_9356_PI426546238 -0.4251523
CUST_8034_PI426546238 -0.33870792
CUST_5048_PI426546248 1.2822595
CUST_8652_PI426544517 0.23535156
CUST_10181_PI426546238 0.10462761
CUST_11936_PI426546248 -0.31739044
CUST_18116_PI426546248 0.22034311
CUST_2689_PI426546248 0.45401192
CUST_1124_PI426544517 -0.17258263
CUST_2294_PI426547332 -0.05142784
CUST_11934_PI426546248 -0.5893712
CUST_14249_PI426544517 0.27462697
CUST_11899_PI426544517 0.03383732
CUST_1975_PI426546238 0.110910416
CUST_4969_PI426546238 0.11335373
CUST_8559_PI426546238 -0.2100339
CUST_8457_PI426544517 -0.012857437

Total number of rows: 48221

Table truncated, full table size 1578 Kbytes.




Supplementary file Size Download File type/resource
GSM1289596_253514710015_201307230417_S01_GE1_107_Sep09_2_3.txt.gz 8.9 Mb (ftp)(http) TXT
Processed data included within Sample table

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