The F2 generation was raised to the age of 3 months (2.17 ± 0.02 cm, 0.42 ± 0.01 g) before use in the cold acclamation experiment. 13°C was selected to represent cold stress. Cold stress was applied for 2 h according to previous study and our own preliminary experiments. Before cold treatment, the fish were transferred to tanks with small openings in the bottom and allowed to stand for one day. They were gently moved to the cold bath for the experiment. The groups (n = 30 per group) were designated m3ck-13°C, m3ck-28°C, wt-13°C, and wt-28°C. After temperature stress for 2 h, the fish were anesthetized with 100 mg/L MS222 and transferred immediately into liquid nitrogen.
Growth protocol
Wild-type and transgenic fish were reared at 28°C ± 0.5°C and under a photoperiod regime of 14/10 h light/dark. Water quality was maintained by circulation with a filtration system.
Extracted molecule
total RNA
Extraction protocol
RNA samples were extracted from whole zebrafish with Trizol Reagent (Invitrogen, CA, USA). RNA quality as expressed by the RNA integrity number (RIN) was determined on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and then purified with an RNeasy mini kit (QIAGEN GmBH, Germany) with the RNase-Free DNase Set (QIAGEN).
Label
Cy3
Label protocol
Total RNA was amplified and labeled with Cy3-CTP by Low Input Quick Amp Labeling Kit, One-Color (Agilent technologies, Santa Clara, CA, USA), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany).
Hybridization protocol
Each slide was hybridized with 1.65 µg Cy3-labeled cRNA using the Gene Expression Hybridization Kit (Agilent technologies Santa Clara, USA) in Hybridization Oven (Agilent technologies Santa Clara, USA). After 17 hours hybridization, slides were washed with Gene Expression Wash Buffer Kit (Agilent technologies Santa Clara, USA).
Scan protocol
Slides were scanned using Agilent Microarray Scanner (Agilent technologies Santa C lara, USA) with default settings, Dye channel: Green, Scan resolution=5 μm, PMT 100%, 10%, 16 bit.
Description
Gene expression at low temperature in wild type fish
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, USA). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, USA).
