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•Genes showing stable expression in barley after transfer to low-nitrogen conditions.•UBE2, EF2 and β-TUB6 selected from RNAseq data.•Optimal number of reference genes for qRT-PCR normalisation calculated as two.•Combination of β-TUB6 and GAPDH2 recommended for qRT-PCR normalisation.•Selection of reference genes based on RNAseq and qRT-PCR data shown to be valid.
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