RT-qPCR Analysis of Seed and Seedling Transcripts

Total RNA was isolated from whole imbibed seeds or seedlings and further analysed by RT-qPCR. RT-qPCR was performed in a Roche LightCycler 480 and the SensiFast^TM SYBR^® No-ROX Kit (Bioline), using standard PCR conditions according to the manufacturer’s instructions. The cDNA used for RT-qPCR was prepared from 2 µg of total RNA (prepared with the LiCl method) using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific). We used about 20 ng of cDNA per RT-qPCR sample in 10 µl reactions. Parallel reactions were used to normalize the amount of template cDNA (Merchan et al., 2007 (link)); normalized expression was calculated using the mean expression of three to four control transcripts. The specific primers for the four genes used for transcript abundance normalization [Ntubc2, L25, EF-1α (Schmidt and Delaney, 2010 (link)) and NtPsbA] are included in Supplementary Table S1 available at JXB online. Specificity was confirmed by analyses of the RT-qPCR profiles. Reproducibility of RT-qPCR was achieved by running technical duplicates, and by using two independent cDNA preparations. At least two biological replicates were performed per condition. Transcript levels of COP1, HSP26, PsaG, PsbR, POR, HY5, PHYA1, and PHYB1 genes were analysed using the specific primers also listed in Supplementary Table S1.