Porcine Stem Cell Gene Expression

Porcine OCT4, CDX2, or NANOG gene expression levels were analyzed by real-time quantitative (q)PCR using the ΔΔCT method[28 (link)]. Total RNA was extracted from 50 oocytes using a Dynabead mRNA DIRECT Kit (Life Technologies; Foster City, CA, USA). First-strand cDNA synthesis was completed using a cDNA Synthesis Kit (Takara; Kyoto, Japan) and oligo(dT) 12–18 primers. The PCR primers used to amplify OCT4, CDX2, and NANOG genes are listed in S1 Table. Real-time PCR was performed with SYBR Green in a final reaction volume of 20 μL (qPCR kit, Finnzymes; Vantaa, Finland). PCR conditions were as follows: 95°C for 3 min, followed by 39 cycles of 95°C for 15 s, 57°C for 15 s, 72°C for 45 s, and a final extension of 72°C for 5 min. Finally, relative gene expression levels were quantified by normalizing to the respective GAPDH mRNA level. Experiments were conducted in triplicate.