We used the protocol illustrated in Ojo et al. to perform PCR intensification for the evaluation of genes whose primers (Primer3 software) are listed below [24 (link), 44 (link)]. In a 25 μl volume mixture of 2 μl cDNA (10 ng), 2 μl primer (100 pmol), and 2 μl water, PCR enhancement was achieved. 12.5 μl Ready Mix Taq PCR master mix (One Taq Quick-Load 2x, master mix, NEB, Cat: M0486S) and 8.5 μl nuclease-free water. An initial denaturation at 95°C for 5 minutes was followed by 20 cycles of amplification (denaturation at 95°C for 30 seconds, annealing for 30 seconds, and extension at 72°C for 60 seconds) and a final extension at 72°C for 10 minutes). In all tests, we incorporated negative controls, where the mixture has no cDNA. The amplicons were sorted out on a 1.5% agarose gel (Cleaver Scientific Limited: Lot: 14170811) in Tris (RGT reagent, China, Lot: 20170605). -Borate (JHD chemicals, China, Lot 20141117) EDTA buffer (pH 8.4). The primer information can be found in the S1 File .
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