RNA Isolation and qRT-PCR Analysis of N. furzeri

RNA isolation from N. furzeri embryos and tissues using TRI reagent (Sigma-Aldrich) and quantitative Real Time RT-PCR analysis were performed as described (Zupkovitz et al., 2018b (link)). Total RNA was isolated from either snap-frozen whole embryos at different stages or brain, liver and muscle samples of adult fish following the manufacturer’s instructions. One microgram of isolated RNA was reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad) and Real-time RT-PCRs were performed using the CFX384 Well qPCR Detection System (Bio-Rad) and Blue S’Green qPCR Kit (Biozym). TATA binding protein (tbp) housekeeping gene expression was used for normalization. All used primer sequences are listed in Supplementary Table 3. All expression raw values obtained from the Real-Time RT-PCR analysis (normalized to tbp) are listed in Supplementary Table 4. Real-time PCR experiments were initially analyzed with the CFX Maestro software. Graphical output and statistics were performed with Prism software (GraphPad). Statistical significances were calculated with one-way ANOVA implementing Tukey’s multiple comparisons. p-values were calculated employing Prism software and standard deviation (SD) is shown. p > 0.05; *: p ≤ 0.05; **p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.