Real-time PCR Gene Expression Analysis

Real-time PCR was performed using the LightCycler 96 Roche Real-time PCR system and PowerUp™ SYBR™ Green Master Mix (applied biosystem by Thermo Fisher Scientific). Seventeen differentially expressed genes (from shoots and roots of both low N and high N) were selected for validation. Primer3 version 2.4.0 was used to design gene-specific primers and their specificity was verified using the NCBI database through the Blast tool (Supplementary Table S5). The 10 µl RT-qPCR reaction contained 1 µl of template cDNA (20 ng), 1 µl of forward primer, 1 µl of reverse primer, 4 µl of PowerUp™ SYBR™ Green Master Mix, and 3 µl of H₂O. PCR was run at an initial denaturation of 94°C for 3 min followed by 40 cycles of 94°C for 10 s, 60°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min to check the specificity of amplification. The housekeeping gene TaATP (ATP-dependent 26S proteasome regulatory) (Paolacci et al., 2009 (link)) was used as the endogenous control and all reactions were performed in triplicate. Relative gene expression was analyzed using the 2−^ΔΔCt method.