Real-time quantitative polymerase chain reaction

mRNA extraction from oocytes at the MII stage and in vitro-cultured embryos at day 6 (20 embryos per sample, three replicates) by a Dynabeads mRNA Direct Kit (DynalAsa, Oslo, Norway). SuperScript™ III reverse transcriptase (Invitrogen, Grand Island, NY, USA) and oligo (dT)12–18 primers were used to synthesize first-strand cDNA. Real-time quantitative PCR was conducted on a Step One Plus Real-time PCR system (Applied Biosystems, Warrington, UK) using the primers specified in Table 1. The total reaction volume for the final PCR consisted of 20 μL, including SYBR Green PCR Master Mix (Applied Biosystems). As follows were the amplification conditions: 10 min at 94°C, followed by 39 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 55°C, and extension for 55 sec at 72°C, and a final extension for 5 min at 72°C. Relative mRNA expression levels were determined according to the 2^−∆∆Ct protocol [28] by normalization to GAPDH.

PCR, polymerase chain reaction; bp, base pair; F, forward; R, reverse.