RNA Extraction and qPCR Analysis of Whole Grains

Total RNA was extracted from samples comprising two whole grains using the Sigma-Aldrich Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St Louis, MO) with the addition of a 6-min incubation with thermostable α-amylase (Megazyme, Wicklow, Ireland) in lysis buffer at room temperature prior to addition of β-mercaptoethanol (Betts et al., 2017 (link)). Following treatment with TURBO DNase-free (Ambion, Life Technologies, Waltham MA), cDNA synthesis was performed using SuperScript®III Reverse Transcriptase according to manufacturer's instructions (Life Technologies, Waltham, MA). Details of gene names, MLOCs and primer details are presented in Table S1. QPCR primers were designed using Primer 3 software (Koressaar and Remm, 2007 (link)) and selected based on specificity as determined by blastn software (Table S1; Acland et al., 2013 (link)). qPCR was performed as described by Burton et al. (2008 (link)) with data normalised using the reference genes HvCyclophilin, HvGAPdH2, HvHSP70, and HvTubulin (Vandesompele et al., 2002 (link)).