4.9. Real-Time PCR (RT-PCR) Based Gene Expression Quantification

Total RNA was isolated from HepG2 after treatment using the RNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The cDNA synthesis was done using the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA samples were mixed with the PowerUp SYBR Green Master Mix (Applied Biosystems, Foster city, CA, USA) and RT-PCR was performed using qTOWER³ G (Analytik-Jena, Jena, Germany). The RT-PCR cycling parameters included an initial denaturation of 95 °C for 10 min and a primer extension at 95 °C for 15 s and 60 °C for one minute with 40 cycles. Ct values were obtained, and relative expression 2^(−ΔΔCt) was calculated and analyzed for changes in mRNA expression. Primers used for the study are mentioned in Table 1. Peptidylprolyl Isomerase A (PPIA) gene was used as a housekeeping gene.

Primers used for the analysis of gene expression.