Quantitative PCR Analysis of Zebrafish Gene Expression

Total mRNA was extracted from 30 larvae using Trizol (Invitrogen) and reverse-transcribed using SuperScript® III First-Strand (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed in triplicated using a Rotor-gene Q (Qiagen) and 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne), following the manufacturers’ protocols. The cycling parameters were 95 °C for 14 min, followed by 45 cycles at 95 °C for 15 s, 59 °C for 20 s and 72 °C for 20 s. Threshold cycles (Ct) and dissociation curves were generated automatically by Rotor-gene Q series software. Sample Ct values were normalized with Ct values from zebrafish actb2 and rplp0, used as control genes. Primer sequences are listed in Supplementary Table 1. All primers were designed using the software Primer3^{19 (link)}.