Quantification of Gene Expression by RT-qPCR

Total RNA was isolated from the roots and shoots of plants subjected or not to HM treatments as previously reported [37 (link)]. RT-qPCR analysis was performed using SYBR Green fluorescent detection in a CFX96 Real-Time System Cycler (Bio-Rad, Hercules, California, United States) with three biological and three technical replicates per sample. The primer sequences used are reported in Table 1. The PCR program was as follows: 10 min at 95 °C, 50 cycles of 15 s at 95 °C, 20 s at 60 °C, and an increment of 0.5 °C every 0.5 s from 65 °C to 95 °C. The specificity of PCR products was checked in a melting curve test. Differences in gene expression between treated and untreated samples were considered significant when the expression was at least doubled (greater than or equal to two-fold upregulation) or halved (less than or equal to two-fold downregulation) according to [43 (link)].