Bioscript reverse transcriptase kit

The head and thorax from individual stabilized tissue was transferred into a plastic 2 ml screw-cap vial containing 500 µl of TRIsure RNA extraction reagent (Bioline, London, UK). The tissue was homogenized by adding two 3 mm glass beads and shaking the samples for 1.5 min using a Mini-Beadbeater-96 (BioSpec Products, Bartlesville, OK, USA) sample homogenizer. Total RNA was extracted according to the manufacturer's directions for TRIsure, using 100 µl of amylene-stabilized chloroform, 250 µl of isopropyl alcohol and two washes of 1.0 ml 75% ethanol. Total RNA pellets were eluted in 20 µl of RNase-free water. A standard quantity of 350 ng of total RNA per sample was treated to remove genomic DNA contamination using DNase I amplification grade DNase (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, and reverse transcribed to cDNA using the BioScript reverse transcriptase kit (Bioline). The BioScript protocol was followed with the following modifications: reactions were primed using 0.5 µg custom synthesized anchored oligo-DT₂₀ and then incubated with 100 U BioScript and 20 U of RNAse Inhibitor (Bioline) at 45°C for 60 min followed by 70°C for 10 min. The resulting cDNA was diluted 5-fold.

Total RNA was extracted from skin samples of 10 fish per diet from both Trial#1 and #2 using TRI reagent (Sigma), as instructed by the manufacturer. RNA was also extracted in the same manner for the Trial#1 gut samples (n = 6). The concentration and quality of the RNA was assessed by both the ND-1000 Nanodrop Spectrophotometer (Labtech Inc., East Sussex, UK) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Cheshire, UK). RNA (2 μg) was used for cDNA synthesis using Bioscript reverse transcriptase kit (Bioline, London, UK) and 0.2 μg of Random Hexamer Primer (Thermo Scientific, Northumberland, UK). The mixture was incubated at 70°C for 5 minutes and then left to rest on ice for 2 minutes. The master mix for 25 μL reactions was prepared as described in the kit protocol. The tubes were incubated at 25°C for 10 minutes, 45°C for 60 minutes and 72°C for 10 minutes. The cDNA was diluted five-fold using 1x Tris-EDTA (TE) buffer (Sigma).