Expression analysis of apoptotic-related genes

The expression of apoptosis-related genes, caspase-3 (CASP3) and caspase-9 (CASP9) were analyzed using real-time PCR according to Fard et al. (22). The cell lines were treated with each extract and controlled at established IC₅₀ concentration. Thereafter, RNA was isolated using the Trizol reagent according to manufacturer instructions. The concentration and purity of the RNA samples were verified on an agarose gel and by the A260/A280 ratio using a NanoDrop Spectrophotometer. RT-qPCR was performed by the use of SYBR^® Green dye (One-Step RT-PCR Kit, Bio-Rad) on a Rotor-Gene Q real-time PCR cycler. The sequences of used primers for amplification are as follows:

Caspase3-F 5′-TTC ATT ATT CAG GCC TGC CGA GG-3′ and Caspase3-R 5′-TTC TGA CAG GCC ATG TCA TCC TCA-3′, Caspase 9 F 5′-CGAACTAACAGGCAAGCAGC-3′, and Caspase 9 R 5′-ACCTCACCAAATCCTCCAGAAC-3′ and GAPDH F 5′-GTCTCCTCTGACTTCAA-3′ and GAPDH R 5′-ACCACCCTGTTGCTGTA-3′.

The PCR cycling conditions for caspases-9 and -3 were as follows: amplification at 95°C for 1 min, denaturation for 30 cycles at 95°C for 15 s, annealing at 56.6°C, and 57°C for GAPDH for 15 s, primer extension at 72°C for 10 s, and final extension at 72°C for 7 min. Additionally, the quantitative analysis measured the threshold cycle (CT) values in exponential phase amplification, and ΔCT was calculated from the difference between CT values of Casp3, Caspase9, and GAPDH gene. Then, 2^–ΔΔCt was calculated from the fold changes of treated cells in correlation with untreated cells.

Where, ΔCTE is the difference between the CT values of the Casp3, Cas9, and the CT is the value of the GAPDH gene of the treated cells.