Fig. 7

Validation of RNA-seq (rnaseq) data by RT-qPCR (qpcr) in control (2n and 3n) and EE2-treated diploid (2n + EE) and triploid (3n + EE2) zebrafish gonads. (A). Validation of differentially expressed genes (DEGs) in EE2-treated diploid zebrafish gonads (diploid EE2) compared to -diploid control. (B) EE2-treated triploid zebrafish gonads (triploid EE2) compared to the triploid control. The genes subjected to validation were the same for both group comparisons, and included hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (hsd3b1); cytochrome P450, family 17, subfamily A, polypeptide 1 (cyp17a1); cytochrome P450, family 11, subfamily C, polypeptide 1 (cyp11c1); collagen, type I, alpha 2 (col1a2); gonadal somatic cell derived factor (gsdf); forkhead box O3b (foxo3b); suppressor of cytokine signaling 3a (socs3a); anti-Müllerian hormone (amh); aldehyde dehydrogenase 1 family member A2 (aldh1a2); cytochrome P450, family 26, subfamily b, polypeptide 1 (cyp26b1); and vitellogenin 1 (vtg1). The S18 subunit was used as a reference gene to normalize the expression of our target genes. Log2 fold changes for individual genes between groups were calculated independently from bulk RNA-seq and RT-qPCR data, and the agreement between methods was evaluated using Spearman correlation, with 95 % confidence intervals displayed. Gene expression trends were consistent between both methods, showing a strong positive correlation (Pearson correlation coefficient, PCC = 0.90 and 0.99; p = 0.000023 and 0.000001 for A and B, respectively), confirming the reliability of the transcriptomic data.