2.6. RT-PCR Program and Amplification System

The reverse transcribed cDNA was used for the RT-PCR analysis. The volume of the amplification system of RT-PCR was 20 μL, including 10 μL of 2 × PCR Master Mix (Biomed, Beijing, China), 0.5 μL of 10 mmol/L primer (forward and reverse), 1.0 μL of cDNA, and 8 μL of ddH₂O. The amplification program was as follows: initial denaturation at 95 °C for 5 min; followed by the optimal cycles (34, 34, and 36 cycles each for BMPR1B, BMP15, and GDF9) of denaturation at 95 °C for 30 s (see Figure 1); annealing for 30 s; and extension at 72 °C for 60 s, with a final extension at 72 °C for 5 min, and then the PCR product was stored at 4 °C.

Amplification results of BMPR1B, BMP15, and GDF9 in different reaction cycles. M: DL2000 DNA marker.