4.4. RNA Extraction and cDNA Synthesis

For RNA extractions, 9 fish per sex and condition were used. Liver samples were homogenised using Precellys^®24 (Bertin Technologies, Montigny-le-Bretonneux, France) in Trizol reagent (Invitrogen, Carlsbad, CA, USA) with 2.8 mm ceramic beads for 2 × 20 s, separated by 15 s of break, at 5500 rpm. Luciferase control RNA (Promega), 10 pg per 1.9 mg of tissue, was added to each sample to allow for data normalisation. Total RNA was then extracted according to the instructions of the manufacturer of Trizol reagent. Total RNA obtained (2 µg) was then reverse transcribed to cDNA in duplicate using the SuperScript III RNase H-Reverse Transcriptase kit (Invitrogen) with random primers (Promega, Charbonnières-les-Bains, France).