Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions

The EF1 alpha genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1 alpha gene family (named EF1 alpha 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1 beta and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1 alpha genes were the most stable in cotyledons (EF1 alpha 3 and EF1 alpha 1b), epicotyls (EF1 alpha 1a2, EF1 alpha 2b and EF1 alpha 1a1), hypocotyls (EF1 alpha 1a1 and EF1 beta), pods (EF1 alpha 2a and EF1 alpha 2b) and roots (EF1 alpha 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1 alpha genes was found; however, some genes were relatively stable in leaves (EF1 alpha 1a2) and roots (EF1 alpha 1a1) treated with SA as well as in roots treated with PEG (EF1 alpha 2b). EF1 alpha 2a was the most stably expressed EF1 alpha gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1 alpha genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.