Real-time quantitative PCR analysis of Tomato stress response

To extract RNA from shoots and roots, 3 weeks old control and treated plants were harvested at 2pm in the greenhouse. Total RNA was extracted using the AccuPrep® Universal RNA extraction kit (Bioneer, Daejeon, Korea) and treated with RNase-free DNase to remove DNA fragments (Qiagen, Hilden, Germany). One microgram of total RNA was used to synthesize cDNA with AccuPower® RT PreMix (Bioneer, Daejeon, Korea). Quantitative real-time RT-PCR was conducted using a T100TM Thermocycler system (Bio-Rad, Hercules, CA, USA). Primer information is given in Table S7. Reactions (10 µL final volume) were prepared using 5 µL of LaboPass™ SYBR Green Q Master Kit (Cosmogenetech, Dajeon, Korea). 0.5 pmol of a primer pair, and 0.5 µL of cDNA template. 4 biological samples and two technical replicates were used for the quantifications. Ubiquitin was used as the reference. gene expression analysis was performed with the 2^−ΔΔCt method using Bio-Rad CFX Maestro software v.4.0 (Bio-Rad). Baseline and threshold levels were set according to the manufacturer’s instructions. qPCR gene expression profiling. Soot and root samples were acquired and cDNA synthezised using 1microgram RNA. qPCR was conducted using CYBR green with Biorad C-1000.