Oligo dt 12 18 nucleotide primers

Real-time quantitative PCR and the ΔΔC_T method normalized by β-actin were employed to measure the KIF2A gene expression. Each sample contained 50 oocytes, and total RNA was extracted by a Dynabead mRNA DIRECT kit (Life Technologies AS, Oslo, Norway). A cDNA synthesis kit (Toyobo, Osaka, Japan) was used to generate the first strand cDNA with Oligo (dT) 12–18 nucleotide primers (Takara Bio Inc., Tokyo, Japan). The following primers were used to amplify the KIF2A and β-actin cDNA fragments:
KIF2A forward, 5′-GACCTGGCTGGGAACGAAAG-3′
KIF2A reverse, 5′- TTTGTTTCTACCTAAGGCTCGGATG-3′
β-actin forward, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′
β-actin reverse, 5′- ATGGAGCCACCGATCCACA-3′
The SYBR Green real-time PCR Master Mix kits (Life Technologies, Carlsbad, CA, USA) was utilized with a Step One real-time PCR system (Applied Biosystems, Foster City, CA, USA). The following conditions were used to perform the process: 50 °C for 2 min and 95 °C for 2 min; followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.

Analysis of Axin1 gene mRNA expression was measured by real-time quantitative PCR and the ΔΔC_T method. We used a Dynabead mRNA DIRECT kit (Life Technologies AS, Oslo, Norway) to extract the total RNA from 70 oocytes. First strand cDNA was generated using a cDNA synthesis kit (Toyobo, Tokyo, Japan), using Oligo(dT) 12–18 nucleotide primers (Takara Bio Inc., Tokyo, Japan). A cDNA fragment of Axin1 was amplified using the following primers: forward, CAC CCA GAA GCT GCT ATT GGA GA, reverse, CCA GGG CAT AGC CAG AGT TGA. We used SYBR Green real-time PCR Master Mix kits (Life Technologies, Carlsbad, USA) with a Step One real-time PCR system (Applied Biosystems, Foster City, USA) under the following conditions: 50°C for 2 min and 95°C for 2 min; followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.