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.

紫花苜蓿(L.) 莱德布。因其在我国北方牧草生产和生态修复方面的价值而备受关注。感兴趣基因的基因表达谱可以为生物学功能研究提供准确可靠的结果，除了mRNA之外，包括作为参与各种生物学功能的关键元素的微小RNA（miRNA）。定量实时聚合酶链反应 (RT-qPCR) 是一种有用的方法，已广泛用于 mRNA 和 miRNA 的基因表达分析，选择始终稳定的参考基因对于在不同实验条件下标准化 RT-qPCR 数据至关重要. 在本研究中，使用 DeltaCt、Bestkeeper、NormFinder、geNorm 和 RefFinder 方法评估了九个候选参考基因的表达稳定性。在不同的实验条件下，内参基因的表达稳定性不同。全面的，EF-1α作为推荐用于标准化的不同子集中更稳定的参考基因。分析GN4和miR156f的相对表达水平以检验最佳参考基因的可靠性。结果表明，最好的稳定参考基因可以同时用于通过 RT-qPCR 分析对 mRNA 和 miRNA 表达进行标准化，这可能有助于对该豆科植物的遗传研究。