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
B6 (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.
