Quantitative Real-Time PCR Analysis

The TRIzol reagent (Ambion, Cartsbad, CA, USA) was used to extract the cells and isolate the total RNA. The superscript III preamplification system (Invitrogen, Carlsbad, CA, USA) was used to synthesize the cDNA. TaqMan^Tm gene expression master mix and PCR FAM dye-labeled probes for human MT2A (Hs01591333_g1). HO-1 (Hs00157965_m1), β-actin (Hs01060665_g1), and 18S (Hs03003631_g1) were from Applied Biosystems (Foster City, CA, USA). The CFX Connect Real-Time PCR system (Bio-Rad Laboratories, Foster city, CA, USA) was used to perform the quantitative real-time PCR as described previously [23 (link)]. The mean cycle threshold (Ct) values for the 18S or β-actin control probe were used in the normalization of target gene expression. All the reactions were carried out in triplicate, and each experiment was performed at least 3 times independently.