Quantifying PI3K/Akt/mTOR Pathway Genes

After incubating HepG2 cells at a density of 2×10⁶ cells/4 mL in a 60 mm culture dish for 24 h and exposing them to the extracts for an additional 24 h, total RNA was extracted using E.Z.N.A.® Total RNA Kit I following the manufacturer’s instructions. The RNA contents were analyzed at absorbances of 260 and 280 nm using a spectrophotometer (NanoDropTM One/Onec Microvolume, Thermo Scientific, USA). TetroTM cDNA Synthesis Kit was used for the complementary DNA (cDNA) synthesis from messenger RNA templates at 5 μg/μL for quantitative reverse transcription-polymerase chain reaction for PI3K, Akt, and mTOR following the manufacturer’s instructions. Then, cDNA at 1 ng/μL was used for real-time PCR analysis, which was carried out using 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (no ROX) in a CFX Connect Real-Time PCR System (Bio-rad CFX manager version 3.1) at the following conditions: 40 cycles 95°C for 30 s; 60°C for 30 s; 72°C for 30 s. The relative expression level of genes was quantified and normalized to that of GAPDH as the housekeeping gene using the 2^-ΔΔCt method [22 (link)]. The sequences of the primers (10 pg/μL) are listed in Table 1.