Relative Quantification of Gene Expression

All qPCR assays were previously published (vasa, gsdf, cyp19a1a, amh; Skaftnesmo et al., 2021 (link)). A qPCR reaction was prepared to contain 800 nM of each forward and reverse primer, 250 nM of the probe in a 6 µl reaction containing 1x concentration of the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and 2 µl of a 1/20 diluted cDNA. The reaction was subjected to thermocycling in a QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific) with an initial hold at 50°C for 2 min followed by an initial denaturation step at 95°C for 2 min. Thermocycling was conducted for 40 cycles using a denaturation step at 95°C for 1 s followed by a combined annealing and extension step at 60°C for 30 s. Data was processed at Thermo Fisher cloud using the relative quantification app. QPCR was performed in duplicates in 384-well optical plates in a QuantStudio 5 Real-Time PCR system (ThermoFisher Scientific). No-template controls for each gene were run in all qPCR plates. The relative gene expression level was calculated using the 2−ΔΔCt method. All values were normalized to ef1a and calibrated to the average ΔCt of the controls of each sex.