RT-qPCR Analysis of HMG and FPS Gene Expression

Total RNA was extracted from seedling samples (100 mg fresh weight) using the PureLink RNA Mini Kit (Ambion, Life Technologies) following the manufacturer’s instructions. The RNA samples were treated with DNase I (DNA-free kit; Ambion) in a final reaction volume of 25 µL, and cDNA was synthesized from 1 µg of total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and oligo(dT) primers. Real-time PCR was performed using LightCycler 480 equipment (Roche Diagnostics). The raw PCR data from LightCycler software 1.5.0 were used in the analysis. Amplification curves were analyzed using the second derivative maximum method, and crossing points were determined for each curve. For efficiency determination, a standard curve of six serial dilution points (ranging from 200 to 6.25 ng) was made in triplicate. Specific primer pairs for HMG1 and HMG2 mRNAs were described previously (Nieto et al., 2009). The following specific primer pairs were used for FPS1 and FPS2 mRNAs: qFPS1fw (5′-AAAGTCTCAGCCCTCAAAAATTTC-3′), qFPS1rv (5′-CAAGAATAAAAGTGAGGCAGGTTT-3′), qFPS2fw (5′-CGTTTTATTCTTCTGACATTTATGTAT-3′), and qFPS2rv (5′-AATCTCAAATTCTATTTTCGGAAGG-3′). Quantification of transcript levels was done in three independent biological replicates, and for each biological replicate, three technical replicates were performed. PP2AA3 (At1g13320) was used as a housekeeping gene with previously designed oligonucleotides (Hong et al., 2010). The cycle threshold change (ΔCT) was calculated as follows: ΔCT = CT (Target) − CT (PP2A). The fold change value was calculated using the expression 2 − ΔCT (Livak and Schmittgen, 2001).