Gene Expression Analysis by Quantitative Real-Time RT-PCR

Total RNA was extracted from Arabidopsis, tomato, rice, and maize whole seedlings, grown as described above, using the PureLink RNA MiniKit (Ambion) and treated with PureLink DNaseI (Ambion) to remove DNA contamination according to the manufacturers’ instructions. Five-hundred nanograms of RNA was reverse transcribed into cDNA using Superscript II reverse transcriptase (200 units) and oligo(dT) primers (500 ng; Thermo Fisher Scientific), according to the manufacturer’s recommendations. Real-time quantitative RT-PCR (qPCR) was performed in 384 well plates on a 7900HT Fast Real-Time PCR system (Applied Biosystems) using Power SYBR Green master mix (Applied Biosystems) and the following amplification program: 10 min denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The data were analyzed using the comparative cT method (2^−ΔCT) normalized to the following reference genes: GAPDH (At1g13440) and actin B (At3g18780) for Arabidopsis; GAPDH (Os8g03290) and EF1α (Os3g08020) for rice; GAPDH (Sl5g014470) for tomato; and GAPDH (GRMZM2G046804) for maize. Primers used are listed in Supplemental Table S2. Each experiment was performed with three biological and three technical replicates.