RNA Isolation, cDNA Synthesis, and qRT-PCR Analysis

Total RNA was isolated from cells using trizol reagent (Invitrogen, Carlsbad, CA, United States) and RNA purity was determined by NanoDrop spectrophotometer (BioTek, Winooski, VT, United States). Further, 2 μg RNA was used for cDNA synthesis using maxima first cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. Briefly, RNA was treated with Maxima Reverse Transcriptase enzyme and reaction buffer. Thereafter, RT-PCR and analysis were done on ABI 7500 Fast real-time PCR system (Waltham, MA, United States) at cycling conditions as: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 60 s at 72°C. The analysis was done using comparative Ct method. β-actin was used as an internal control (Khan et al., 2016 (link)). Results are normalized to β-actin and depicted as relative expression (fold change). Primers used in the qRT-PCR are as follows: