tissue: red muscle sampling time point: 72hr after injection
Treatment protocol
Fish were injected intraperitoneally with either LPS from Escherichia coli (6 mg/kg body weight; Sigma) or with the same volume of PBS (control group). At the end of the experiment, fish were removed from tanks, anesthetized and the white and red muscles were collected from control and from LPS-injected fish after 24 and 72 hours (n = 5 for each sampling point and treatment), snap frozen in liquid nitrogen and stored at -80 °C.
Growth protocol
Fish were kept in two tanks of 500 L each with a closed seawater circulation system supplied with a continuous flow-through of oxygenated seawater at 18 oC and under controlled photoperiod (12 hours light – 12 hours dark). Fish were maintained and manipulated in accordance with national legislation for the welfare of animals. After two weeks of acclimatization period, fish were randomly divided into two experimental groups. One tank contained twenty untreated fish (control) and the other tank contained twenty lipopolysaccharide (LPS) treated fish. Before injection and sampling, fish were anesthetized using MS222 (0.1 g L-1; Sigma, Alcobendas, Spain) and were fasted 24 hours before the experiment and the sampling period.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. The quantity of the isolated RNA was tested spectrophotometrically, while its quality was tested by electrophoresis in agarose gel (1% W/V). Afterwards, cDNA was synthesized from 5 μg DNase-treated (RQ1 DNase, Promega, Barcelona, Spain) total RNA in a 20 μl reaction, using SuperScript III Transcriptase (Invitrogen) and a mix of oligo(dT) (Promega) and random primers (Promega) according to the manufacturer’s protocols. The RNA was stored at -80ºC until use.
Label
Cy5
Label protocol
Total RNA from pooled control (n = 5) and LPS-treated (n = 5) sea bream white and red muscle tissues was labeled with Cy3-dUTP and Cy5-dUTP (GE Healthcare, Barcelona, Spain) using SuperScript III Transcriptase. The cDNA synthesis reaction was performed at 50ºC for 2 hours in a 20 µl reaction volume, followed with RNA degradation with 0.2 M NaOH at 37ºC for 15 min and alkaline neutralization with 0.6 M Hepes. Labeled cDNA was purified with Microcon YM30 (Millipore, Madrid, Spain).
tissue: red muscle sampling time point: 72hr after injection
Treatment protocol
Fish were injected intraperitoneally with either LPS from Escherichia coli (6 mg/kg body weight; Sigma) or with the same volume of PBS (control group). At the end of the experiment, fish were removed from tanks, anesthetized and the white and red muscles were collected from control and from LPS-injected fish after 24 and 72 hours (n = 5 for each sampling point and treatment), snap frozen in liquid nitrogen and stored at -80 °C.
Growth protocol
Fish were kept in two tanks of 500 L each with a closed seawater circulation system supplied with a continuous flow-through of oxygenated seawater at 18 oC and under controlled photoperiod (12 hours light – 12 hours dark). Fish were maintained and manipulated in accordance with national legislation for the welfare of animals. After two weeks of acclimatization period, fish were randomly divided into two experimental groups. One tank contained twenty untreated fish (control) and the other tank contained twenty lipopolysaccharide (LPS) treated fish. Before injection and sampling, fish were anesthetized using MS222 (0.1 g L-1; Sigma, Alcobendas, Spain) and were fasted 24 hours before the experiment and the sampling period.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. The quantity of the isolated RNA was tested spectrophotometrically, while its quality was tested by electrophoresis in agarose gel (1% W/V). Afterwards, cDNA was synthesized from 5 μg DNase-treated (RQ1 DNase, Promega, Barcelona, Spain) total RNA in a 20 μl reaction, using SuperScript III Transcriptase (Invitrogen) and a mix of oligo(dT) (Promega) and random primers (Promega) according to the manufacturer’s protocols. The RNA was stored at -80ºC until use.
Label
Cy3
Label protocol
Total RNA from pooled control (n = 5) and LPS-treated (n = 5) sea bream white and red muscle tissues was labeled with Cy3-dUTP and Cy5-dUTP (GE Healthcare, Barcelona, Spain) using SuperScript III Transcriptase. The cDNA synthesis reaction was performed at 50ºC for 2 hours in a 20 µl reaction volume, followed with RNA degradation with 0.2 M NaOH at 37ºC for 15 min and alkaline neutralization with 0.6 M Hepes. Labeled cDNA was purified with Microcon YM30 (Millipore, Madrid, Spain).
Hybridization protocol
The slides were pretreated with 1% BSA (fraction V), 5 × SSC, 0.1% SDS (30 min at 50°C) and washed with 2 × SSC (3 min) and 0.2 × SSC (3 min) at room temperature and hybridized overnight at 60ºC in a cocktail containing 1.3 × Denhardt's, 3 × SSC, 0.3% SDS, 2.1 μg/μl polyadenylate and 1 μg/μl yeast tRNA. All chemicals were from Sigma. After hybridization, slides were washed at room temperature in 0.5 x SSC and 0.1% SDS for 15 min, 0.5 x SSC and 0.01% SDS for 15 min, and twice in 0.06 x SSC for 2 and 1 min, respectively.
Scan protocol
Scanning was performed with ScanArray 5000 (GSI Lumonics) and images were processed with GenePix Pro 5.0 (Axon).
Data processing
The measurements in spots were filtered by criteria I/B ≥ 3 and (I-B)/(SI + SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analysis and genes presented with less than three high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Locally weighted non-linear regression (Lowess) normalization was performed, first for the whole slide and next for twelve rows and four columns per slide. To assess differential expression of genes, the normalized log intensity ratios, or difference of the mean Log2 ER from zero, were analyzed for every pair of slides with reverse labeling (6 spot replicates per gene on each slide, n = 12) with Student´s t-test (p < 0.01).
