Evaluation of Housekeeping Gene Expression Stability in Carnation (Dianthus caryophyllus)

Abstract Background: The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability. A suitable reference gene is an absolute prerequisite for accurate normalization, nevertheless, the frequently-used reference gene was reported unstable under different experimental conditions and causes failure to correctly analyze the expression of the interested gene. Therefore, it is vital to systematically evaluate the expression stability of these candidate reference genes before performing RT-qPCR. Results: In this study, two computational statistical methods were used, including geNorm and NormFinder, in order to determine the expression stability of 12 frequently-used reference genes in Dianthus caryophyllus across different experimental conditions. The results show that the expression stability of candidate genes varies greatly in different sample pools, which again proves the instability of these common housekeeping gene expressions. In general, the expression of UBQ10 (ubiquitin10), EF1a (elongation factor 1A) and TIP41 (TIP41-like family protein) were relatively stable under different experimental conditions, while the expression stability of 18S (18S Ribosome RNA), TIF5A (translation initiation factor 5A) and PP2A (protein phosphatase 2A) were relatively poor. Conclusion: EF1α, TIP41 and UBQ10 were considered the most appropriate reference genes when all samples were put together, while UBQ10 was most stable in exogenous hormone treatments. TUB and UBQ10 can be used as reliable internal control genes under stress, while CYP and TUA can act as reliable internal controls in different tissues. This is the first systematic study of selection of reference genes in Carnation, and will benefit future expression studies in this crop.