Table 2.

Cycling conditions for RT-quantitative PCR (qPCR).

Gene	Initial denaturation		Cycling conditions1			Initial melt temp (°C)	No. of cycles
	Temp (°C)	Time (min)	Denaturation	Annealing	Extension
Hypothalamus
GnRH-I	95	10	95°C for 20 s	62°C for 20 s	72°C for 20 s	62	50
GnIH	95	10	95°C for 20 s	60°C for 30 s	72°C for 20 s	60	40
OPN5	95	10	95°C for 15 s	59°C for 30 s	72°C for 20 s	59	45
VA-Opsin	95	10	95°C for 15 s	60°C for 30 s	72°C for 20 s	60	45
Pituitary gland
FSH beta-subunit	95	10	95°C for 15 s	60°C for 20 s	72°C for 20 s	60	45
LH beta-subunit	94	10	94°C for 20 s	60°C for 20 s	72°C for 20 s	60	40
GnRH-RI	95	10	95°C for 10 s	57°C for 15 s	72°C for 25 s	57	50
GnRH-RIII	95	10	95°C for 15 s	58°C for 15 s	72°C for 30 s	58	50
GnIH-R	95	10	95°C for 10 s	57°C for 15 s	72°C for 15 s	57	50
Reference genes
ACTB	95	10	95°C for 15 s	57°C for 15 s	72°C for 30 s	57	40
GAPDH	95	10	95°C for 10 s	56°C for 30 s	72°C for 30 s	56	40

Targeted genes include hypothalamic genes; GnRH-I, GnIH, OPN5, and VA-Opsin, and pituitary genes; FSH beta-subunit, LH beta-subunit, GnRH-RI, GGnRH-RIII, and GnIH-R, along with reference genes, GAPDH and ACTB.

¹Time is in seconds (s).