Identification of Reliable Reference Genes for Gene Expression Analysis in Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao under Cold and ABA Stress

This study aimed to find stable reference genes of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Am) to measure the gene expression at a precise scale in the conditions of cold and abscisic acid (ABA) stress. Gene expression measurements usually need a good internal control to be accurate particularly when there is a different environment. Our group assessed 6 candidate reference genes in both root and leaf tissues by applying four computational analysis methods and an integrated ranking tool. It was shown that the stability of the genes was dependent on the stress treatment and tissue type. Roots were the most stable under cold stresses with GAPDH-2 and leaves with UBQ-2. UBQ-12 was the best in roots and CYP-4 most appropriate in leaves under ABA stress. In order to confirm these results, we examined the expression level of chalcone synthase (CHS) gene, that produces flavonoid and responds to stress. The findings validated that the patterns of expression were not stable when unstable reference genes were used but the recommended genes resulted in stable normalization. The research offers precise recommendations when choosing the right reference genes in Am under specified experimental conditions that enhance the validity of any study on gene expression in the analysis and also facilitate further studies on stress resistance systems and secondary metabolism in this useful plant used in medicine.