Quantitative real-time PCR analysis
Total RNA from Col-0, Stw-0, and Or-0 seedlings (1-, 2-, and 3-week-old)
and leaves (4-, 5-, and 6-week-old plants) was isolated and purified
using RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s
instructions. RNA concentration and purity were verified using a
NanoDropTM 1000 Spectrophotometr (Thermo Scientific,
Walthman, MA). RNA was treated with RNase-free DNase I (Thermo
Scientific) according to the manufacturer’s instructions. 160 ng RNA per
each sample was used for first-strand synthesis using
SuperScriptTM II First-Strand Synthesis System for
RT-PCR (Thermo Scientific) and oligo-dT primers according to the
manufacturer’s procedure. 2 µl of cDNA was used for real-time PCR
analysis, using 0.6 µl each of gene-specific primers listed in Table S10
in a total volume of 20 µl of Luminaris HiGreen High ROX qPCR Master Mix
(Thermo Scientific) in a real-time thermal cycler STEPOnePlus (A&B
Applied Biosystems, Waltham, MA) as instructed. Statistical analysis was
performed using Annova with a post-hoc Turkey test.