Cisplatin-Induced DNA Damage Signaling Pathway

The relative mRNA expressions after cisplatin stimulation in IMC-3CR were analyzed using an RT² Profiler^TM PCR array (Human DNA Damage Signaling Pathway, Qiagen) according to the manufacturer’s protocol34 (link). Briefly, IMC-3CR cells (5 × 10⁵) were placed in RPMI 1640 supplemented with 10% heat-inactivated FCS. Cells with or without cisplatin (1 μg/ml) were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 6 h. After incubation, cells were washed with PBS, total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized through RT performed with an RT² First Strand Kit (Qiagen) according to the manufacturer’s protocol. The cDNA was applied to the PCR array and real-time PCR 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 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing for 1 min at 60 °C. The PCR array results were uploaded to the RT² Profiler PCR Array Data Analysis website (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The alteration of mRNA expression was analyzed using ΔΔCt method.