Transcription profiling of environmentally sampled flounders (Platichthys flesus)
Ontology highlight
ABSTRACT:
ORGANISM(S): Platichthys flesus
SUBMITTER: Tim Williams
PROVIDER: E-MTAB-396 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In Sparus aurata, seasonal temperature variations outside the normal thermal range, may trigger physiological responses leading to pathologies and death. In the present study two groups of wild sea bream were exposed for 21 days to two temperature regimes: 16 ± 0.3 °C (control group) and 6.8 ± 0.3 °C (cold-exposed group). Samples were collected during the acute phase (0, 6 and 24 hours after temperature drop) and upon chronic exposure (21 days). A comparative analysis of gene expression was conducted between S. aurata exposed to 6.8°C. Two groups of wild sea breams (120±16 g body weight; 21±1 cm total length) caught in Valle Bonello were kept in duplicate groups in four 5 m3 circular recycling tanks and acclimated over one month at 16 °C (Di Marco et al. 2010). Fish were then exposed for 21 days to two different temperature treatments: 16 ± 0.3 °C (control groups) and 6.8 ± 0.3 °C (cold groups). Both groups were fasted during the trial. Warm and cold water temperature conditions were maintained by supplying tanks with two separate re-circulating systems equipped with mechanical and biological filters. Cold water conditions were achieved by inducing two consecutive temperature drops: water was firstly gradually cooled from 16 °C to 12 °C at a rate of 1°C day -1 and then reduced from 12 °C to 6.8 °C within 24 h. This temperature was then kept for 21 days. Ten fish per treatment were sampled at different intervals during acute: 0 (T1), 6 (T2) and 24 hours (T3), and chronic exposure (21 days; T4) to low temperature. Fish were firstly anaesthetized in a solution of tricaine methanesulphonate (300 mg/l; MS 222, Finquel) in a 30-litre bucket and then euthanized by severing their spinal cord. Liver samples (about 30 mg) were collected from each specimen, incubated in RNA later at 4 °C for 24 h and then stored at -20 °C. Total RNA was extracted from tissue samples using the RNAeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration was determined using a UV-Vis spectrophotometer NanoDrop® ND-1000 (NanoDrop Technologies, Wilmington, USA). RNA integrity and quality was evaluated by means of Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and Expert software according to Ferraresso et al. (2008). RNA integrity number (RIN) index was calculated for each sample that provides a numerical assessment of RNA integrity. This value facilitates the standardization of the quality interpretation; only RNAs with RIN >8 were used for further microarray experiments. Once single RNA samples were extracted and measured, 3 pools (Pool_A consisting of four individuals, Pool_B and _C of three) per each sampling-time point were prepared. This was aimed at getting a sufficient statistical power which is reached by a minimum of three biological replicates per analysed point and at reducing microarray analysis cost. To perform DNA microarray analyses has been used the microarray platform GPL6467 (Ferraresso et al. 2008). Sample labelling and hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol. For each of 24 liver pools 500 ng of total RNA were linearly amplified and labelled with Cy3-dCTP, using the Low RNA Input Linear Amplification Kit PLUS one-color (Agilent Technologies). A total of 24 Agilent 4 x 44K sea bream microarrays (12 samples from both control and cold groups) were then hybridized. An Agilent G2565BA DNA microarray scanner was used to scan arrays at 5 µm resolution, Feature Extraction Software 9.5.1 was then used to process and analyse array images. The software returns a series of spot quality measures in order to evaluate the goodness and the reliability of spot intensity estimates. Filtering and cyclic lowess normalization were performed using R statistical software (http://www.r-project.org/).
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