RT-qPCR Quantification of Gene Expression

Total RNA from desired tissues was isolated using the RNeasy kit (Qiagen, MD, USA). The cDNA was prepared using one µg of total RNA as instructed in the kit protocol (TransGen cDNA preparation kit). The cDNA was then used for RT-qPCR with 2X qPCR superMix (TransGen) in a 20 μL reaction on a Bio-Rad CFX96 Touch™ real-time PCR machine (Bio-Rad, Singapore). The cycling parameters were: 95°C for 30 s; 40 cycles of 95°C for 10 s and 60°C for 15 s. The fold change in the gene expression was determined using the EF1α gene as the internal control using Livak method (2^−ΔΔCT) as described earlier (Livak and Schmittgen, 2001 (link); Aslam et al., 2019 (link); Jakada et al., 2019 (link); Aslam et al., 2020 (link)). Three biological replicates and at least three independent technical replicates were performed in each condition. To analyze statistical significance, a two-tailed Student’s t-test was used *** indicates p < 0.001, ** indicates p < 0.01 and and * indicates p < 0.05. The primers used in this study are listed in the Additional File S2.