Investigating NMD Inhibition Impact on GFP

GFP reporters were agroinfiltrated with the p14 silencing suppressor and either mock or the dominant-negative Upf1 inhibitor of NMD as previously described [25 (link)]. At 5 days post-infiltration, leaves were imaged and collected for RNA extraction using Trizol. Total RNA was treated with RQ1 DNase (Promega) prior to reverse-transcription quantitative PCR (RT-qPCR). Small PCR fragments (<200 bp) were amplified using the SYBR Green-based Luna One-Step RT-qPCR kit according to the manufacturer’s protocol (New England BioLabs). The Roche LightCycler 480 platform was used for all experiments. p14 served as an internal reference gene for relative GFP gene expression using the 2^−∆∆Ct method.