Quantitative RT-PCR for Gene Expression Analysis

For cDNA synthesis, 2 µg of total RNA were reverse transcribed with QuantiNova Reverse Transcription (RT) Kit (Qiagen, Germany). To estimate amounts of cDNA templates of the selected genes, quantitative RT-PCR assays were performed using specific primers listed on Table S1, designed by Primer3Plus (Untergasser et al., 2012 (link)). q-PCR was performed in an Applied Biosystems StepOne real time PCR system. Each 10 µL reaction contained 5 µL of SYBR Green PCR Master mix (2 X), 0.5 µM primers mix and 2 µL of template cDNA (1/10 dilution). The thermocycler was programmed to run for 5 min at 95°C, followed by 40 cycles of 15 s at 94°C, 30 s at 60°C. Transcript accumulation of each gene was normalized relative to the constitutively expressed Elongation Factor 1 B (Glyma.02G276600) gene. Amplification efficiencies of the different primer combinations were all > 90%. Relative expression was determined using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). Each data point is the mean value of three biological replicates. Two technical replicates were used for each sample.