Quantifying Gene Expression in Tomato Leaves

For qRT-PCR analysis of gene expression, leaves from two-week-old tomato plants, untreated or treated with 20 µM or 200 µM DOR, were harvested 3 days after toxin administration. Total RNA was extracted from 100 mg of homogenized leaves using RiboZOL (vWR, Radnor, PA, USA). For cDNA synthesis, 20 μg of total RNA was retro-transcribed by using the FastGene Scriptase II cDNA kit (Nippon Genetics Europe, Düren, Germany), according to the manufacturer’s instructions, and stored at −80 °C until use. qRT-PCR experiments were performed, as previously described [35 (link)], using the LightCycler apparatus (Roche, Basel, Switzerland) and the SYBR GREEN dye (PCR Biosystems, London, UK). The 2^−ΔΔCt method was applied to evaluate the level of gene expression, using the ACT3 and UBI3 genes of S. lycopersicum as housekeeping genes. Results represent mean values ± SD of independent experiments (n = 3). Samples were run in technical triplicates. Statistical significance was attributed by Student’s test (p < 0.05). The primers used for amplification are listed in Supplementary Table S1.