Detection of expression of apoptosis-related genes (cas3 and p53) by quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the ETA-exposed HeLa cells that had been treated with HuscFvs and controls for 12 h, using a GeneJET RNA Purification Kit (Thermo Fisher Scientific). The quality of the RNAs was determined at OD_260nm and OD_280nm by NanoDrop™ 2000/2000c Spectrophotometer. After DNase treatment using RNase-free-DNase I (Thermo Fisher Scientific), the RNAs were used to generate cDNA by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time RT-PCR was performed on KAPA SYBR^® FAST qPCR (Kapa Biosystems, Cape Town, South Africa). Each reaction mixture (20 µl) contained 10 µl of 2 × KAPA SYBR^® FAST qPCR Master Mix Universal, 200 nM final concentration each of forward and reverse primers, and 20 ng of cDNA template in nuclease-free PCR-grade water. The reaction was performed in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories). The following thermal cycles was used: initial denaturation at 95 °C for 3 min and 40 cycles at 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s. A dissociation curve was generated from a thermal profile consisting of 95 °C for 1 min, 55 °C for 30 s, and 95 °C (0.5 °C/s). Each sample was amplified in triplicate. Gene expressions relative to normal cells were analyzed using the ΔC_T method. The amounts of casp3 and p53 were normalized to the internal control, i.e., GAPDH. The primers used in these experiments are shown in Supplementary Table ^S2.