Abstract
Radish (Raphanus sativus) is an important cruciferous root crop with a close relationship to Chinese cabbage (Brassica rapa). RT-qPCR is used extensively to evaluate the expression levels of target genes, and accurate measurement of target gene expression with this method is determined by the valid reference genes used for data nomalization in different experimental conditions. Screening for appropriate reference genes with stable expression based on RT-qPCR data is important for gene expression and functional analysis research in radish and its relatives. However, many researches have thought that almost no single reference gene is widely suitable for all experimental conditions, and few researchers have paid attention to the validation of reference genes in radish gene expression analysis. In the present study, 12 candidate reference genes were selected for analysis. Their expression in 28 samples, including 20 radish samples from different organs and conditions, four Chinese cabbage organs and four organs of their distant hybrid, was assessed by RT-qPCR and then five software tools—ΔCt, geNorm, NormFinder, BestKeeper and RefFinder—were used to compare their expression stability. The results showed that the most suitable reference genes were different in different organs and conditions. GAPDH, DSS1, and UP2 were optimal reference genes for gene expression analysis in all organs and conditions in radish. UPR, GSNOR1, and ACTIN2/7 were the most stable reference genes in different radish organs. UP2 and GAPDH were suitable reference genes for radish pistil development studies. RPII, UBC9, and GAPDH had the most stable expression in radish under various stresses. DSS1, UP2, and TEF2 were the optimal reference genes for Chinese cabbage organs, whereas TUA was optimal for the distant hybrid. UP2, and TEF2 were appropriate reference genes for all of the samples together. The optimal reference genes we identified, UP2, GAPDH, UPR, and GSNOR1 were verified by normalizing the expression patterns of YAB3, RPL, and FUL. These results will provide important information for selecting target reference genes in different research contexts and improve the accuracy and precision of gene expression analysis for radish, Chinese cabbage and their distant hybrid.
Highlights
RT-qPCR (Quantitative Real-time PCR) is an important and effective method to evaluate the expression of target genes in different tissues, organs and conditions (Bustin, 2000, 2002; Gachon et al, 2004)
Using primers designed for each gene, the PCR amplification specificities of the 12 candidate genes were checked by 2% agarose gel electrophoresis with pistil cDNA samples from radish, Chinese cabbage and their distant hybrid
The results showed that all 12 candidate genes had specific amplification and the product lengths were consistent with the expected lengths in radish, Chinese cabbage and their distant hybrid (Figure S1)
Summary
RT-qPCR (Quantitative Real-time PCR) is an important and effective method to evaluate the expression of target genes in different tissues, organs and conditions (Bustin, 2000, 2002; Gachon et al, 2004). Expressed reference genes are usually used to normalize transcriptome quantification through exposure to the same preparation processes as the target genes. Appropriate reference genes for data normalization are critical to obtain accurate expression data by RT-qPCR (Ginzinger, 2002; Gachon et al, 2004; Dheda et al, 2005; Guenin et al, 2009; Schmidt and Delaney, 2010). An appropriate reference gene should be expressed stably in different types of cells, tissues and organs, and at different developmental stages. Its expression should be high or moderate and assumed to be unaffected under different experimental conditions (Bustin, 2002; Brunner et al, 2004; Czechowski et al, 2005; Gutierrez et al, 2008)
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