Reference gene selections for real time quantitative PCR analysis of gene expression in different oat tissues and under salt stress

Abstract

Appropriate choice of reference genes for data normalization is of critical importance for accurate real time reverse transcription quantitative PCR (RT-qPCR) analysis of gene expression. Oat is an agriculturally important crop cultivated widely around the world for grain or forage, and appropriate reference genes for reliable gene expression analysis remain to be identified. In this study, we selected nine candidate reference genes based on available oat RNA-seq data. We then conducted a systematic evaluation of the relative expression stability of these genes in different tissues and under salt stress using statistical algorithms, geNorm, NormFinder, and BestKeeper. Our findings reveal that the highest-ranked reference genes for accurate data normalization should be selected according to specific sample subsets. For different tissues, the combination of two reference genes [elongation factor 1-alpha (EF1α) and serine/threonine protein phosphatase 2] was sufficient for accurate normalization. For salt stress treatment, the combination of two reference genes (EF1α and TATA-binding protein) was sufficient for accurate normalization. Moreover, the commonly used reference genes, actin and glyceraldehyde 3-phosphate dehydrogenase, were least suitable for data normalization of oat samples. Expression of a salt stress-inducible transcription factor Avena sativa WRKY2 was investigated to validate the reference genes identified in this study. This is the first systematic study of reference gene selection in cultivated oat and provides guidelines to obtain more accurate RT-qPCR results in this species.