Real-Time PCR Analysis of Apoptosis and Proliferation Markers

We used the ΔΔCt method to determine the expression of mRNA using real-time PCR: ΔΔCT = ΔCT test sample—ΔCT calibrator sample. The reaction was carried out using 48-well plates and Luminaris Color HiGreen reagents qPCR Master Mix (Thermo Fisher Scientific); 100 ng of cDNA was used for each reaction. The following genes were examined: casp3, casp9, cytc, aifm1, pcna, ki-67, and mcm2. The primers used for this procedure are presented in Table 2. rpl13a was used as the reference house-keeping gene [65 (link)]. The reaction conditions were set as specified by the manufacturer. Each sample was analyzed in duplicate. The procedure was conducted using a StepOnePlus™ Real-Time PCR System.