Selection of reliable reference genes for gene expression studies in Rhododendron micranthum Turcz

Selection of reliable reference genes is a prerequisite for accurate normalization of gene expression in quantitative real-time polymerase chain reaction (qRT-PCR). The present study is the first report of a systematic evaluation of potential reference genes in Rhododendron micranthum Turcz. The expression stabilities of 6 candidate reference genes (18S, ACT, CYP, UBQ, TUA, and EF1α) in different tissues and the same tissue at different developmental stages were assessed in Rh. micranthum Turcz, using two analysis software packages, geNorm and NormFinder. The results indicated that EF1α and UBQ were the most stable reference genes for different tissues (leaf, root, stem and flower) at the same developmental stage, whereas 18S should be avoided. During leaf development, EF1α and 18S can be used as the most reliable reference genes, but CYP was the most variable one. During flower development, the most stable genes were CYP and EF1α, while 18S was the least stable one. In order to demonstrate the importance of using suitable reference genes for obtaining an accurate and reliable results in gene expression studies, the expression pattern of an independent target gene, RhMDH gene was analyzed in parallel, the RhMDH showed an expression difference of 58-fold in stem when normalized with the most stable gene (EF1α) or the least stable gene (18S) act as reference, respectively. Our results support that the selection of suitable reference genes is necessary to achieve accurate results in qRT-PCR.

► The first report of a systematic evaluation of reference genes in Rhododendron micranthum Turcz.
► We recommend that EF1α and UBQ as reference genes in various tissues.
► During flower development, CYP and EF1α can be used as reference genes.
► During leaf development, the most stable genes were EF1α and 18S.