Quantifying Gene Expression in Zebrafish Embryos

Total RNA was isolated from 15 wild-type zebrafish embryos of different development stages using TRIzol reagent (TaKaRa, Japan). cDNA was synthesized by using the RevertAid Fist Strand cDNA Synthesis kit (Thermo Fisher Scientific, United States). qRT-PCR was performed using SYBR Green (TaKaRa, Otsu, Japan) on a QuantStudio Dx Real-Time PCR Instrument with QuantStudio Dx Software (Applied Biosystems, Foster City, CA, United States). The 18s ribosomal RNA (18-s) gene was chosen as the reference gene (McCurley and Callard, 2008 (link)). The relative expression levels of each sample were calculated using the RQ formula (RQ = 2^–ΔΔCq) (Bustin et al., 2009 (link)); and these assays were carried out in three independent triplicates, with final calculations based on the means of triplicate wells. The sequences of primers are shown in Supplementary Table 1.