Quantitative PCR for Plant Gene Expression

Quantitative PCR was performed as previously described (Øverby et al., 2015 (link)). Briefly, fresh plant material was snap-freezed in liquid nitrogen and subsequently grounded from which total RNA was extracted using Spectrum Plant Total RNA Kit. RNase-Free DNase Set was used to prevent DNA contamination. RNA concentration was measured using NanoDrop 1000 (Thermo Scientific). cDNA was synthesized using QuantiTect Reverse Transcription Kit, and qPCR was performed with SYBRgreen in 96-well plate in a Lightcycler 480 (Roche Applied Science) with the following program: preincubation step (95°C, 5 min), 45 amplification cycles (95°C, 10 s; 55°C, 10 s; 72°C, 10 s), and a final melting curve analysis. Cycle threshold values, PCR efficiencies and relative expression values were calculated using Lightcycler 480 Software (Roche), LinRegPCR and REST 2009 (QIAGEN), respectively. At4g24550 (Clathrin) and At4g34270 (TIP41-like) were used as housekeeping genes. Primer sequences are given in Supplementary Table S1.