Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress

Abstract

Background: Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five 'traditional' RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database. Results: Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, Ref-Finder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition. Conclusions: Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

RNA-Seq Time Series of Vitis vinifera Bud Development Reveals Correlation of Expression Patterns with the Local Temperature Profile

“…Additionally, novel candidate genes were identified which displayed a constant expression level. This aligns well with a previous study, which reported that novel reference genes identified by genome-wide in silico analysis outperformed typical reference genes in common wheat [34].…”

RNA-Seq Time Series ofVitis viniferaBud Development Reveals Correlation of Expression Patterns with the Local Temperature Profile

“…Additionally, novel candidate genes were identified which displayed a constant expression level. This aligns well with a previous study, which reported that novel reference genes identified by genome-wide in silico analysis outperformed typical reference genes in common wheat [32].…”

Reference gene selection for real‐time quantitative PCR assays in different tissues of Huperzia serrata based on full‐length transcriptome sequencing

“…We had to screen the most suitable RGs for studying the HupA‐biosynthesis. Based on the CPM value of transcriptome sequencing and reported literatures (Chen et al, 2019 ; Dudziak et al, 2020 ; Wu et al, 2020 ), 10 traditional and three new candidate RGs were chosen (Table 1 ) . Especially for three new candidate RGs, they had stable expression in full‐length transcriptome sequencing but have not been used as RGs before.…”