A reliable result obtained by qRT-PCR highly depends on accurate transcript normalization using stably expressed reference genes. However, the transcript levels of traditional reference genes are not always stable. Also, the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated in Arabidopsis under
iron deficiency. Our results revealed that three novel reference genes (
SAND, YLS8 and TIP41-like) were identified and validated as suitable reference genes for qRT-PCR normalization in both
iron deprivation (the addition of
Ferrozine to the medium) and
starvation (withdrawal of
iron from the medium) conditions. This conclusion was also confirmed by publicly available microarray data. In addition, when using
SAND, YLS8 and TIP41-like as multiple reference genes, the expression patterns of FIT1 and IRT1, two
iron deficiency marker genes, were approximately similar with that reported previously. However, a weaker inducible response was obtained from qRT-PCR by normalizating EF-1α alone. Together, we proposed that the combination of
SAND, YLS8 and TIP41-like can be used for accurate normalization of gene expression in
iron deficiency research. These results provide a valuable evidence for the importance of adequate reference genes in qRT-PCR normalization, insisting on the use of appropriate reference gene validation in all transcriptional analyses.