The total RNA (2 ig) was reverse transcribed into single-stranded cDNA using the oligo(dT) primer and SuperScriptTM first-strand synthesis system for RT-PCR (Invitrogen). Each RNA sample was treated with RNase-free DNase I (Invitrogen) to remove any genomic DNA contamination. The integrity of all RNA samples was verified by the successful amplification of 18S RNA. The following gene-specific primers (Table 1) were used for PCR detection: P5 and P6 for grouper gnrh, P7 and P8 for grouper cyp19a1a, P9 and P10 for grouper cyp19a1b, P1 and P2 for grouper cga, P3 and P4 for grouper Ihb, and 18SU and 18SD for the 18S RNA as a positive control in the RT-PCR.
The cycling conditions were as follows: 95°C for 4 min; 35, 33, 33, 30, and 30 cycles of denaturation at 94°C for 30 sec; 56°C, 55°C, 55°C, 64°C, and 58°C for 30 sec for gnrh, cyp19a1a, cyp19a1b, cga, and Ihb, respectively; and 72°C for 1 min; followed by a final extension at 72°C for 7 min. The RT-PCR products were separated on agarose gels, followed by ethidium bromide staining, and then detected under ultraviolet light with Gel Doc 2000 (BioRAD). The cDNA band densities were measured using Quantity One software (BioRAD).