Quantifying mRNA Expression in COCs

mRNA was extracted from 35 COCs after 24 h of culture in IVM medium using a Dynabeads mRNA Direct Kit (61,012; Thermo Fisher Scientific, Waltham, MA, United States), and cDNA was synthesized using the First Strand Synthesis Kit (cat# 6210; LeGene, San Diego, CA, United States) in accordance with the manufacturer’s instructions. The primer sequences for amplification of cDNA were the same as those used in previous studies (Han et al., 2016 (link); Park et al., 2018 (link)). qRT-PCR was performed using a WizPure qPCR Master (W1731-8; Wizbio Solutions, Seongnam, South Korea) according to the manufacturer’s instructions, on a QuantStudio™ six Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, United States). The PCR conditions were as follows: initial denaturation at 95°C for 10°min, followed by 40 cycles of amplification at 95°C for 15°s, 60°C for 20°s, and 72°C for 15°s, and a final extension at 95°C for 15°s. Relative gene expression was calculated using the ∆∆CT method. The primers used are listed in Table 1.