4.5. Real-Time Polymerase Chain Reaction (PCR) Quantification

Total RNA was isolated using Trizol reagent and reverse transcribed into complementary DNA (cDNA) with High-Capacity cDNA Reverse Transcription Kit according to the manufacturer’s instructions. cDNA was synthesized using 2 µg of total RNA in 25 µL reaction mixture. Real-time quantitative RT-PCR assay was performed using the Applied Biosystems FAST 7500 Real-Time PCR system. All reactions were performed in triplicates using the SYBR Green Real-Time PCR master mix containing RNase inhibitor and primers. The primer sequences are shown in Table S1. All PCR data were analyzed using the delta–delta Ct method and normalized to housekeeping gene GAPDH with Applied Biosystems 7500 system SDS software. All samples were compared to their respective controls in each experiment.