Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions

Abstract

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

“…EF1α is a commonly used reference gene and was shown to be one of the best reference genes for ABA in Populus euphraticarice [8]. Additionally, EF1α was selected as best choice for Petunia hybrida [48], soybean [49] and B. brizantha [50]. This indicates that the reference gene verified in Metasequoia is identical to previously reported reference genes.…”

Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina

“…EIF4a , ACTIN 2 , CAC , TUA5 , HISTONE H3 , HSP81 . 2/90 and SAND were chosen because they were already established as reference genes in other species [13–17,19,20]. In addition, we considered PSB33 , ATPase , THIOREDOXIN , HCF and NdhO .…”

“…The eight reference gene candidates EIF4a , ACTIN 2 , CAC , TUA5 , HISTONE H3 , HSP81 . 2/90 , 18srRNA and SAND were chosen due to their stable expression in other species [13–17]. PSB33 , ATPase , THIOREDOXIN , HCF and NdhO were chosen because of their robust expression levels in Arabis alpina in a time-course RNAseq experiment (data provided by Eva Willing, MPIPZ, Cologne) and/or due to their essential function for the plant.…”

Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]

“…Therefore, it is prudent to experimentally validate the appropriateness of reference genes in the target species rather than its universally acceptance across species [ 37 , 38 ]. A number of evaluation approaches have been adopted in plant species to verify these inconsistencies in expression of conventional reference genes through systematic studies in Arabidopsis [ 35 ], potato [ 39 ], barley [ 40 ], sorghum [ 21 ], Setaria viridis [ 41 ]; melon [ 42 ], pearl millet [ 20 ], goose grass [ 43 ], foxtail millet [ 44 ], soybean [ 45 ] and ryegrass [ 46 ].…”