4.4. Quantitative RT-PCR

Total RNA was isolated from 30 homogenized zebrafish embryos using 1 mL of TRIzol reagent (Molecular Research Center Inc., Cincinnati, OH, USA). Chloroform was added for protein separation and isopropanol was added for the precipitation of RNA. Reverse transcription of 3 µg total RNA was performed using SuperScriptF IV Reverse transcriptase (Thermo Fisher, Waltham, Massachusetts, MA, USA). qRT-PCR analysis was performed using PowerUp SYBR green Master Mix (Thermo Fisher). Expression of target genes was analyzed by the comparative threshold method and the results were normalized to β-actin as an endogenous control gene. qRT-PCR data represents data obtained using biological and technical triplicates.