The expression of Bcl2, Survivin, cIAP, XIAP, GCLC (glutamate-cysteine ligase catalytic subunit) and GAPDH was analyzed in MCF-7 and U87 cells after the treatment with PRE, EA, Hex and mixture of EC+GA+UA. Total RNA was extracted from cells using RNAeasy kit (Qiagen GmbH, Hilden Germany). cDNAs were reverse transcribed from 1 μg of total RNA from each sample using Quantiscript Reverse Transcriptase, Quantiscript-RT-buffer and RT-primer-mix of QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden Germany) according to the manufacturer’s protocol.
Amplification of cDNA was carried out in 20 μl solution containing 2 μl cDNA, 10 pmol primer pairs for Bcl2, Survivin, XIAP, cIAP and GAPDH (for primer sequences- Table A inS1 File ) and 10 μl of RT qPCR Master mix (Qiagen GmbH, Hilden, Germany). The PCR consisted of initial denaturation at 94°C for 5 min, followed by 25 reaction cycles (30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C) and a final cycle at 72°C for 10 min. GAPDH was used as internal control. The amplified PCR products were separated by agarose gel electrophoresis and visualized with ethydium bromide. The abundance of each target mRNA was detected and normalized to that of GAPDH mRNA.
Amplification of cDNA was carried out in 20 μl solution containing 2 μl cDNA, 10 pmol primer pairs for Bcl2, Survivin, XIAP, cIAP and GAPDH (for primer sequences- Table A in