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

Abstract

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.

Identification of Suitable Reference Genes for Gene Expression Normalization in Jatropha curcas L During Development and Under Stress Conditions Using Real Time Quantitative PCR

“…The present studyshowed robust results, and revealed the best reference genes suggested by the GeNorm, NormFinder and BestKeeper to obtain reliable results for qPCR normalization. It should be pointed out that transcriptomes could differ considerably among specific seed tissues.This fact has been shown in soybean (Le et al 2007, Saraiva et al, 2014, barley (Barrero et al 2009), Arabidopsis (Le et al 2010). Therefore, the stably expressed genes validated in this study in tissues of developing seedlings and under various stress conditions can be used in future studiesunder the same employed conditions.…”

Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli

“…RAN was also the optimal performer in Cucumis melo (Kong et al, 2014) treated with growth regulators but was variable under different conditions in the present study. EF1 α was previously reported as the most stably expressed reference gene in many species, for example, Glycine max under drought and salinity stresses (Saraiva et al, 2014), Solanum tuberosum under abiotic stresses (Nicot et al, 2005), Saccharum spp. under drought and salinity stresses (Guo et al, 2014), Pennisetum glaucum under individual and multiple abiotic stresses (Shivhare and Lata, 2016).…”

Stacked genetically modified soybean harboring herbicide resistance and insecticide rCry1Ac shows strong defense and redox homeostasis disturbance after glyphosate-based herbicide application

“…Hence, each treatment contained three biological pools of three different plants, which were used for the transcriptomic analysis ( Figure 1). [29], F-box [30], 60S [31], 18SrRNA [32] and Skip16 [33]. Normalization of cycle threshold (Ct) values was performed using the 60S ribosomal protein housekeeping gene as it showed low variability (<15%) and high e ciency results (98,59%; R2 = 0.99).…”