Abstract
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.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) is one of the most powerful and reliable techniques to quantify gene expression and is widely used because of its sensitivity, accuracy and reproducibility in gene expression analysis
Reference genes are generally housekeeping genes that are universally expressed in all cells and whose products are necessary for cytoarchitecture or basic metabolism (Bustin, 2002; Xu et al, 2011), such as Actin, Tubulin, EF1-α, and GAPDH
In Salicornia europaea, the α-tubulin gene has been used as an internal control to quantify target gene expression under salt stress (Lv et al, 2011; Ma et al, 2013); the stability of α-tubulin in S. europaea was not verified in this previous study
Summary
Real-time quantitative polymerase chain reaction (RT-qPCR) is one of the most powerful and reliable techniques to quantify gene expression and is widely used because of its sensitivity, accuracy and reproducibility in gene expression analysis. Reference genes are generally housekeeping genes that are universally expressed in all cells and whose products are necessary for cytoarchitecture or basic metabolism (Bustin, 2002; Xu et al, 2011), such as Actin, Tubulin, EF1-α, and GAPDH. These traditional reference genes are not always stably expressed in different cases (Glare et al, 2002; Everaert et al, 2011; Migocka and Papierniak, 2011). To the best of our knowledge, there is no previous report on the selection of suitable reference genes for S. europaea
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