2.3. Total RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)

2 × 10⁵ cells/mL HaCaT cells were seeded onto 12-well cell culture plates overnight (n = 3) and treated with PG102 at designated concentrations at different time points. Treatment with PG102 at 0.25~2.0 mg/mL did not cause cytotoxicity in HaCaT cells [15]. Total RNA was isolated from cells using RNAiso (Takara Bio, Shiga, Japan) according to the manufacturer's protocol, followed by complementary DNA (cDNA) synthesis using AMV reverse transcriptase (Takara Bio) and oligo dT primers (Qiagen, Valencia, CA). Real-time quantitative PCR (RT-qPCR) of each cDNA was performed using SYBR Premix Ex Taq™ (Takara Bio) and Thermal Cycler Dice Real Time System (Takara Bio) with the primer pairs listed in Table 1. The mRNA levels were normalized by the level of HPRT, and the relative changes in gene expression to untreated controls were calculated by the 2-^ΔΔCt method.

List of primers used for RT-qPCR.