Abstract
BackgroundA suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya (Hylocereus) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya.ResultsIn this study, six candidate reference genes i.e. Actin(1), GAPDH, UBC(1), UBC(2) EF1-α(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1-α(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong (Hylocereus monacanthus) and Guanhuabai (H. undatus) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data.ConclusionsIn this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species.
Highlights
A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR analyses
Results from BLAST analyses demonstrated that 27 reference genes had maximum identity (80–92%) with similar deduced polypeptides from Chenopodium quinoa, 7 reference genes had maximum identity (93 to 99%) with similar deduced polypeptides from H. monacanthus, Cycle threshold (Ct) value analyses of six reference genes Ct values of Actin(1), eukaryotic elongation factor 1-alpha (EF1-α)(1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin-conjugating enzyme (UBC)(1), UBC(2) and histone(1) were calculated to determine their transcript levels in different tissues, temperature stresses, fruit developmental stages of Guanhuahong and Guanhuabai pitayas
To the best of our knowledge, this study is the first report on systematically evaluating the expression stability of different potential reference genes for quantitative real-time PCR (qRT-PCR) in the Cactaceae family
Summary
A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. Quantitative real-time PCR (qRT-PCR) is a nucleic acid quantitative technology to study gene expression level in molecular biology research [1]. It combines the sensitivity of conventional PCR with a cost-effective assay using a specific fluorescent signal. The accuracy of gene expression levels using qRT-PCR depends on the use of stable reference genes to normalize the difference between samples [6]. It is necessary to select the most suitable reference gene for accurate qRTPCR evaluation according to different species and experimental conditions [16, 17]. Calculations of reference genes by geNorm [6], NormFinder [18] and BestKeeper [19] is essential for normalization of qRTPCR analyses
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