Quantitative Real-Time PCR for Housekeeping Genes

Quantitative real-time PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad) and run in triplicate in 48-well plates with an MJ Mini Thermal Cycler (Bio-Rad). The cycling profile consisted of 95°C for 1 min, followed by 40 cycles of 5 s at 95°C and 20 s at 60°C, as recommended by the manufacturer. The amplification process was immediately followed by a melting curve analysis steps of 0.5°C every 10 s from 65°C to 95°C. Baseline and threshold cycles (Ct) were automatically determined using the Bio-Rad CFX manager software (Bio-Rad). Here, we selected a classical housekeeping gene, Actin, and a novel housekeeping gene, CAC [37] (link) (Table1) as internal controls for constitutive expression in various tissues.