Quantitative Gene Expression Analysis

Total RNA was isolated from three cotyledons with the TRI RNA Isolation Reagent (Sigma-Aldrich) and treated with TURBO DNase (Ambion). Reverse-transcription and RT-qPCR analyses were performed as described in Garcia-Alcazar et al. (2017) (link). RT-qPCR amplifications were performed on a 7300 Real-Time PCR System (Applied Biosystems) and each quantitation consisted of three biological replicates, each with three technical replicates (except for the RNAi lines, which consisted of a single biological replicate). The UBIQUITINE3 housekeeping gene was used as an internal control and the absence of genomic DNA contaminating the RNA sample analyzed by RT-PCR was tested as previously described (Gimenez et al., 2010 (link)). Statistical analyses were performed using Student’s t-tests.