Analyzing Cisplatin-Induced DNA Damage Response

The relative mRNA expressions of genes involved in DNA damage activities were analyzed following cisplatin stimulation of A375 using an RT² ProfilerTM PCR array (Human DNA Damage Signaling Pathway, Qiagen) in adherence to manufacturer’s instructions^{36 (link)}. In brief, cells treated with or without cisplatin (4 μM) were incubated at 37 °C under a humidified atmosphere containing 5% CO₂. After incubation, washing cells with PBS, total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized by RT-PCR using an RT² First Strand Kit (Qiagen) according to the manufacturer’s protocol. The cDNA was applied to the Profiler PCR array and real-time PCR was performed on a Sequence Detection System (ABI PRISM^® 7000; Life Technologies Corporation) using PCR master mix (SA Biosciences RT² qPCR Master Mix; Qiagen) for SYBR Green detection for each reaction. Samples were amplified with a precycling hold at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing for 1 min at 60 °C. Changes in mRNA expression were analyzed using ∆∆Ct method. The expression levels were quantified relative to the values obtained for some housekeeping genes (ACTB, B2M, GAPDH, HPRT1, and RPL13A).