Kod sybr qpcr mix

RNA samples of 12 piglets used for the RNA-seq experiment were analyzed by quantitative real-time PCR (RT-qPCR). cDNA synthesis was performed using ReverTra Ace reverse transcriptase (Toyobo Co., Osaka, Japan) following the manufacturer’s instruction. RT-qPCR was performed using a StepOne Plus real-time PCR system with SYBR Green master mix (Applied Biosystems, Singapore). Specific primers were designed according to the sequences of selected transcripts using the NCBI (Supplemental Table S1). Each real-time RT-PCR reaction in 20 μL of reaction mixture comprised 10 μL of KOD SYBR^® qPCR Mix (TaKaRa, Dalian, China), 0.4 μL of 50 × ROX reference dye, 0.2 μL of each primer, 2 μL of cDNA, and 7.2 μL of RNase-free dH₂O. Swine 18S rRNA was used as an internal control gene, and all samples were analyzed in triplicate. The relative expression levels of genes were determined using 2^−ΔΔCt value methods. All data were analyzed by Student’s t-test using SPSS version 21.0 statistical software. RT-qPCR data were presented as the mean ± standard deviation, and p < 0.05 was considered statistically significant.

Freshly frozen tissues (≤30 mm³) were homogenized using the MagNA Lyser Instrument (Roche, Basel, Switzerland; 03358968001) and MagNA Lyser Green Beads (Roche; 03358941001). Total RNA from freshly frozen tissues and endometrioid adenocarcinoma cells was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany; 74104), and complementary DNA was synthesized from genomic DNA-purified RNAs using qPCR-RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan; FSQ-301). The mRNA expression was measured using KOD SYBR qPCR Mix (TaKaRa Bio, Shiga, Japan; QKD-201X5) and QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific; A40425). Relative gene expression was analyzed using the 2^−ΔΔCt method. The primer sequences for RT-qPCR are listed in Table S2. The experiment was performed in triplicate [24 (link)].