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

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.

Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED

“…Reference genes are identified as the most stable genes exhibited the lowest SD, and genes with SD less than 1 were considered acceptable [66]. As shown in Table 5, β-tub was the most stably expressed genes with the lowest SD values in all samples, short-term heat exposure and the control samples (0.69, 0.58 and 0.24, respectively).…”

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus

“…In chordate phylum, many studies on identification of optimal RGs for gene expression normalization mainly focused on vertebrates, such as mammals, birds, fish and amphibian13141516, but information in amphioxus was limited. Several traditional RGs ( 18   S , ACT , EF1A , GAPDH , TUB and UBC ) have been widely used in qRT-PCR analyses in amphioxus, insects, human and plant17181920, but expression level of these RGs were variable to some degree under different conditions221. Wang et al firstly evaluated the reliability of candidate RGs and found EF1A was a useful RG in different tissues of amphioxus, but number of candidate RGs and algorithms were few in the study (four candidate RGs and two statistical methods)9.…”

Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions

“…3.1 Characterization of the expression profiling of candidate reference genes Gene selection was based on similar studies conducted in other plant species subjected to abiotic stresses, including salinity (Goulao et al 2012;Kandu et al 2013;Guo et al 2014;Xiao et al 2015). The ten candidates [Ubi (Ubiquitin), Tub (Tubulin), S23 (40S ribosomal protein), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), Elf-4a (Eukaryotic initiation factor 4α), Ef-1 (Elongation factor 1α), Ap47 (Clathrin adaptor complexes subunit), Apt (Adenine phosphoribosyltransferase), Act (Actin) and PP2A (Serine threonine protein phosphatase)] complied with the PCR requirements showing a single peak in the dissociation curve and efficiencies ranging between 84 and 104 % ( Table 1).…”