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Real-time quantitative polymerase chain reaction (RT-qPCR), a reliable technique for quantifying gene expression, requires stable reference genes to normalize its data.  Salicornia europaea , a stem succulent halophyte with remarkable salt resistance and high capacity for ion accumulation, has not been investigated with regards to the selection of appropriate reference genes for RT-qPCR. In this study, the expression of 11 candidate reference genes,  GAPDH  ( Glyceraldehyde 3-phosphate dehydrogenase ),  Actin ,  α-Tub  ( α-tubulin ),  β-Tub  ( β-tubulin ),  EF1-α  ( Elongation factor 1-α ),  UBC  ( Ubiquitin-conjugating enzyme ),  UBQ  ( Polyubiquitin ),  CYP  ( Cyclophilin ),  TIP41  ( TIP41-like protein ),  CAC  ( Clathrin adaptor complexes ), and  DNAJ  ( DnaJ-like protein ), was analyzed in  S. europaea  samples, which were classified into groups according to various abiotic stresses (NaCl, nitrogen, drought, cold and heat), tissues and ages. Three commonly used software programs (geNorm, NormFinder and BestKeeper) were applied to evaluate the stability of gene expression, and comprehensive ranks of stability were generated by aggregate analysis. The results show that the relatively stable genes for each group are the following: (1)  CAC  and  UBC  for whole samples; (2)  CAC  and  UBC  for NaCl stress; (3)  Actin  and  α-Tub  for nitrogen treatment; (4)  Actin  and  GAPDH  for drought stress; (5)  α-Tub  and  UBC  for cold stress; (6)  TIP41  and  DNAJ  for heat stress; (7)  UBC  and  UBQ  for different tissues; and (8)  UBC  and  Actin  for various developmental stages. These genes were validated by comparing transcriptome profiles. Using two stable reference genes was recommended in the normalization of RT-qPCR data. This study identifies optimal reference genes for RT-qPCR in  S. europaea , which will benefit gene expression analysis under these conditions.

Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR