Cdna preparation kit

After the RNA isolation from the desired plant sample, TransGen cDNA preparation kit was used to prepare cDNA using one μg of total RNA. The qRT-PCR was carried out using 2X qPCR superMix (TransGen) in 20 μL reaction volumes using Bio-Rad CFX96 Touch™ real-time PCR machine (Bio-Rad, Singapore). The reaction conditions for qRT-PCR included the following steps: 2 min at 95 °C followed by 40 cycles of denaturation for 10 s at 95 °C and annealing for 15 s at 60 °C, and extension for 15 s at 72 °C. At least three biological replicates were used for each experiment with three technical replicates. The fold change in the expression of genes was determined using the Livak method (2^−ΔΔC_T), and the pineapple ef1α gene was used as the internal control [50 (link)]. The primers used in this study are listed in additional Table S7.

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.