tissue: liver agent: control group time: T3 (9 days)
Treatment protocol
An exposure laboratory experiment was performed in the “Ur Biologia eta Experimentazio Zerbitzua” (UBEZ) of the University of the Basque Country (UPV/EHU) A total of 84 eels, 20.23 ± 2.84 cm in length, were acclimatized to laboratory conditions for 2 weeks in a 10 L water glass tanks under controlled conditions: 12 h light/dark cycle at 18oC, conductivity 600 μS. During this period animals were fed with commercial flakes. Ammonium, nitrites and nitrates kits (Sera GmbH) were used to systematically asses the levels of nitrogenous compounds which were maintained at 0-0.5mg/l; 0-0.5 mg/l, and 5-10 mg/l, respectively. When highest concentrations within those ranges were achieved seawater was partially replaced. A day before exposure experiment, animals were placed in 4 aquaria with 8 L of water at 600 μS. In each aquarium 21 eels were randomly distributed and exposed to: metal-mercury (Hg) (Fluka-Sigma-Aldrich) at a concentration of 100μg/L; and to a phitosterol, β-sitosterol (Sigma) at a concentration of 150 μg/ L. The last compound needed ethanol (0.01%) as vehicle. In addition to an ethanol control group, a water control (600μS) without contaminant was also prepared.
Extracted molecule
total RNA
Extraction protocol
Liver (100 mg) was gridded in TRizol® (Invotrgen) using Precellys homogenizer (Bertin technologies, France) and Precellys lysing kit MK28-R (Bertin; 2*5000, 30sec). After that, RNA was extracted following TRIzol® (Invitrogen) manufacturers´s instructions and purified using RNAsy Kit (Qiagen) with the additional DNAse treatment (Qiagen) on columns. RNA quantity and quality was measured using Epoch TM Take 3 TM spectrophotometer (Biotek) and 2100 bioanalyzer (Agilent) respectively. RIN values above 8 were discharged.
Label
Cy3
Label protocol
The hybridization step was performed using the One-Color Microarray-Based Gene Expression Analysis “Low Input Quick Amp Labeling” protocol (Agilent, version 6.5, May, 2010) following manufacturer’s instructions. Total RNA was used as starting material to produce cRNA, which was in vitro synthesized using QuickAmp Labelling Kit one-color (Agilent). At first step, it was retrotranscribed using AffinityScript reverse transcriptase, an MMLV reverse transcriptase and Oligo dT primers coupled to T7 promoter. After that, in vitro transcription and Cy-3 labelling of ds cDNA was performed using the antisense T7 Promoter Primer, T7 RNA Polymerase and Cy-3-CTP.
Hybridization protocol
All cRNA labelled samples purified and quantified using RNeasy spin columns (Qiagen) and NanoDrop ND-1000 spectophotometer respectively. As a result yield and specific activity of each reaction was measured and all of them were above following limits 0.825 ug yield and 6 pmol Cy3 per μg of cRNA. Finally, 50 ng of labelled cRNA were obtained. Hybridization step was performed in a SureHyb hybridization chamber (Agilent), a total of 600ng of labelled cRNA was used in a volume of 40 μL. They were incubated in the hybridization oven at 10 rpm, 65 oC for 17 hours. Slides were washed using GE wash buffer 1 (1 minute), GE wash buffer 2 (37 oC, 1 minute), and acetonitrile wash (10 seconds). Finally, scanning process was performed on a G2565 DNA microarray scanner (Agilent) with ozone barrier slide cover using following settings: Green channel dye, 61*21.6 mm scan region, scan resolution of 3 μm and 20 bit Tiff.
Scan protocol
Feature Extraction software v. 10.7.3.1 (Agilent) was used to feature signal intensity extraction. It was subsequently processed on GeneSpring GX 12.6 (Agilent Technologies).
Data processing
As preprocessing, first all Feature Extraction flags were transformed to Absent/Marginal/Present calls (Compromized/Not Detected/Detected Flags in the new GeneSpring versions), as follows: Feature is NOT Positive and significant: ABSENT (Compromized);Feature is NOT uniform: ABSENT (Compromized);Feature is NOT well above background: MARGINAL (Not Detected);Feature is Saturated: MARGINAL (Not Detected); Feature is Population outlier: ABSENT (Compromized);Threshold raw signals (lineal data) to 1. After that a geometric mean of replicated probes was calculated and the resulted Raw Signal was transformed as Log2. Furthermore, an intra-chip normalization was done based on quantile normalization. Finally a baseline was performed with the mean value of control samples.
