RT-qPCR Validation of RNA-seq Data

Reverse transcription followed by quantitative real-time PCR (RT-qPCR) was performed as previously described [61 (link)] for validating the RNA-seq data. Reverse transcription and PCR of standard curve and individual samples was run with the Gene Amp 9700 PCR machine (Applied Biosystems, Foster City, USA). Real-time RT-qPCR was performed starting with a 5 min template incubation and denaturation step at 95°C, followed by 45 cycles divided in 10 s denaturation at 95°C, 10 s annealing at 60°C and 10 s synthesis at 72°C using the CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, USA) with the LightCycler^® 480 SYBR Green I Master kit (Roche Applied Science, Penzberg, Germany). Samples were amplified in triplicates and the mean was used for further calculations. Normalised expression of target genes was determined using the geNorm algorithm [62 ] based on the geometric mean of 3 reference genes: eef1a1I1 (Eukaryotic translation elongation factor 1 alpha 1, like 1) [34 (link)], tuba1c (Tubulin, alpha 1c) [63 (link)] and actb1 (Actin, beta 1) (S2 File). We investigated gene transcription for fasn (Fatty acid synthase), mat1a (Methionine adenosyltransferase I, alpha), cbsb (Cystathionine-beta-synthase b) and vtg5 (Vitellogenin 5) that were selected among the differentially expressed genes in F₁ livers from the RNA-seq analysis.