4.5. Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR)

The AR-positive PCa cell lines (LNCaP and 22RV1) were seeded into 6-well plates at a density of 2 × 10⁵ cells/well, and then AR antagonist (proxalutamide or enzalutamide) or vehicle (0.1% DMSO) was used to treat the cells. Total RNA was extracted from these treated cells using a High Pure RNA Isolation Kit (RNAiso Plus, Takara, Japan), and then reversely converted into cDNA using a PrimeScript™ RT Reagent Kit (Takara Bio, Japan). The qPCR analysis was conducted using SYBR Premix Ex Taq™ (Takara Bio, Japan) and a CFX96 real-time PCR detection system (Bio-Rad, CA, USA). The gene-specific primers (shown in Supplementary Table S1) were synthesized by Invitrogen (Carlsbad, CA, USA). The cycling conditions of the qPCR program were as follows: 95 °C for 1 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Three replicates were prepared for each measurement. Melting curve analysis was performed to verify the specificity of real-time PCR products. Relative quantification was performed using the 2^−∆∆Ct method, and values were normalized to the reference gene β-actin.