Evaluation of reference genes for normalization of mRNA and microRNA expression by RT-qPCR under different experimental conditions in Medicago ruthenica (L.) Ledeb.

Medicago ruthenica (L.) Ledeb. has received considerable attention due to its value for forage production and ecological restoration in northern China. Gene expression profiles of interesting genes may provide accurate and reliable results for biological function research, in addition to mRNA, including microRNA (miRNA) as a key element that participates in various biological functions. Quantitative real-time polymerase chain reaction (RT-qPCR) is a useful method that has been widely used in gene expression analysis of mRNA and miRNA, and the selection of consistently stable reference genes is essential to normalize RT-qPCR data under different experimental conditions. In this study, the expression stability of nine candidate reference genes was evaluated with DeltaCt, Bestkeeper, NormFinder, geNorm and RefFinder methods. The expression stability of reference genes varied under different experimental conditions. Overall, EF-1α as much more stably reference gene in different subsets recommended for normalization. The relative expression levels of GN4 and miR156f were analyzed to test the reliability of the optimal reference genes. The results suggested that the best stable reference genes can be simultaneously utilized in the normalization of both mRNA and miRNA expression by RT-qPCR analysis, which may facilitate genetic studies of this leguminous plant.