Transcriptomic analysis of seed development

RNA was isolated from dry seeds (0 HAI) as well as from the developing embryos/seedlings at 4, 12, and 24 HAI. The embryos and seedlings were isolated from the endosperm using tweezers, then frozen in liquid nitrogen and stored at −80 °C. For each extraction, approx. 100 embryos and seedlings that were ground in liquid nitrogen and a NucleoSpin RNA Plant and Fungi Kit (Macherey-Nagel, Duren, Germany) were used to isolate the total RNA. The RNA was purified using Qiagen RNase-Free DNase set (Qiagen, Venlo, Netherlands) and an RNeasy MinElute Cleanup Kit (Qiagen, Venlo, Netherlands). The concentration and quality of the isolated RNA was evaluated using an ND-1000 NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was produced using a Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). The primers that were relevant to the genes that were studied are listed in Table 2. The real-time PCR as well as the calculation of the relative expression level were done according to Betekhtin et al. [47 (link)].