Plant Cell Reports

, Volume 33, Issue 9, pp 1453–1465 | Cite as

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

  • Kátia D. C. Saraiva
  • Dirce Fernandes de Melo
  • Vanessa D. Morais
  • Ilka M. Vasconcelos
  • José H. CostaEmail author
Original Paper


Key Message

The EF1α 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 EF1α gene family (named EF1α 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, EF1β 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 EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 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 EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.


Expression stability Glycine max Polyethylene glycol Reference genes Salicylic acid 



Abscisic acid


ADP-ribosylation factor


Cycle threshold


Elongation factor 1α


Elongation factor 1β


Eukaryotic translation initiation factor




Metalloprotease, Insulin degrading enzyme


Naphthylacetic acid


Polyethylene glycol


Salicylic acid


16 SKP1/Ask-Interacting Protein 16


Melting temperature


Hypothetical protein


Untranslated region



This research was supported by CAPES, CNPq and FUNCAP.

Conflict of interest

The authors declare that have no conflict of interest.

Supplementary material

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Kátia D. C. Saraiva
    • 1
  • Dirce Fernandes de Melo
    • 1
  • Vanessa D. Morais
    • 1
  • Ilka M. Vasconcelos
    • 1
  • José H. Costa
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyFederal University of CearaFortalezaBrazil

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