Identifying internal control genes by quantitative real-time PCR for accurate normalisation of gene expression in Gardenia jasminoides
2021
Mu, Zejing | Du, Xiaolang | Wang, Xiaoyun
Gardenia jasminoides J. Ellis. is widely used in horticulture and its fruit is heavily used in the natural pigment and traditional Chinese medicinal industries. Functional discovery and characterisation of genes in fruits generally require accurate transcript quantification. Therefore, we first identified twenty candidate genes and then set out to determine their stability in qRT-PCR assays using three software programs, i.e. geNorm (version 3.5), NormFinder (version 20), and RefFinder. The optimal number of ICGs required for qRT-PCR data normalisation was also determined. Our results indicated that two ICGs were sufficient for accurate qRT-PCR data normalisation in the organs tested. From the consensus genes of the three software programs, EIF3G and GAPDH were the top-ranked ICGs in pulp and seeds at three developmental stages, i.e. the green fruit stage, the orange fruit stage, and the red fruit stage. DEAD and 40S-S15a were the top-ranked ICGs in pulp, leaves, seeds, and stems at the red fruit stage. The reliability of the selected ICGs was confirmed through expression profile comparison of two genes, i.e. phytoene synthase and β-ring hydroxylase involved in carotenoid biosynthesis. This is the first report of ICG screening in G. jasminoides. Our results provide a basis for normalising gene expression levels and understanding of gene function in G. jasminoides.
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