SYBR Green-Based RT-PCR for Gene Expression

The QuantiNova SYBR Green RT-PCR Kit was used for SYBR Green-based real-time amplification (Qiagen Inc., Venlo, the Netherlands). A 20 mL reaction using 10 mL of QuantiNova SYBR Green RT-PCR Master Mix, 1 mL of each forward and reverse primer (Table 1), 6 mL of nuclease-free water, and 2 mL of cDNA was set up. The thermal profile was 30 min at 50 °C, 15 min at 95 °C, 45 cycles of 15 s at 94 °C, 30 s at Tm, and 30 s at 72 °C, followed by a melting curve from 60 °C to 90 °C. All real-time PCRs were performed using a Rotor-Gene Q Real-Time PCR equipment (Qiagen). For gene expression investigations employing OCT 3/4 and SOX 2, triplicate reactions were conducted, and the mean expression value was determined for the following analyses. The (2-ddct) technique was used to compute the relative expression levels of the genes [23 (link)].