Real-time PCR

We extracted total RNA from frozen liver tissue using TRIzol reagent and Direct-zol™ RNA MiniPrep (Zymo Research; Orange, CA, USA) according to the manufacturer's protocol, including DNA digestion step. We checked RNA integrity by 1.5% agarose gel electrophoresis, then determined the RNA concentration (ng/µl) and percentage purity (%) spectrophotometrically with SYNERGY/HTX multi-mode plate reader (BioTek Instruments Inc, USA). We performed reverse transcription using the qScript cDNA synthesis kit (Quantabio Reagent Technologies; QIAGEN Beverly Inc., USA) in a 20 μl final volume containing 5 × cDNA supermix, 200 ng RNA template and distilled water, using PCRmax Alpha thermal cycler (Cole-Parmer Instrument Co. Ltd., UK). Conditions consisted of reverse transcription 25 °C for 5 min, 42 °C for 30 min and 85 °C for 5 min. We designed intron-spanning forward and reverse primers for quail using the Oligo 7 software and checked for target identity using Primer-Blast software of the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov, supplementary material Table S2). We performed quantitative PCR in Agilent AriaMx Real-time PCR System (Agilent Technologies, USA) and applied 5 × HOT FIREPol^® EvaGreen^® qPCR Mix Plus (Solis BioDyne; Tartu, Estonia), 2 ng cDNA template, 200 nM of each primer, and distilled water in a 10 μl final volume of each sample. We ran the samples in duplicates.

We normalised relative changes in gene expression against RPL19 gene expression as the most stabile reference gene selected from 6 housekeeping genes such as ACTB, GAPDH, RPL19, RPS8, 18S, and RPL13 by 3 algorithms (delta Ct, Best Keeper, NormFinder) (Simon et al. 2018; Vitorino Carvalho et al. 2019). Relative gene expression compares the expression level of gene of interest or the target gene to the expression level of the reference gene housekeeping genes that are usually expressed relatively constant in all cells and most of the conditions (Joshi et al. 2022). We collected raw data with Aria AgilentMx 1.8 software. We calculated relative gene expression of the target genes (mTOR, RPS6K1, and IGF1) in fold change using the double-ΔCT method (Schmittgen and Livak 2008). We considered the sample with the highest delta Ct value as calibrator to calculate the double-ΔCT value (Pabinger et al. 2014).