Evaluation of housekeeping gene expression stability in carnation (Dianthus caryophyllus)

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. Suitable reference gene is an absolute prerequisite for accurate normalisation. In this study, three computational statistical methods before performing RT-qPCR, including geNorm, NormFinder and BestKeeper was used to integrated expression stability evaluations of 12 frequently-used reference genes in Dianthus caryophyllus across different experimental conditions. The results showed that the expression stability of candidate genes varies greatly in different sample pools. The expression of TIP41 (TIP41-like family protein) and UBQ10 (ubiquitin10) was relatively stable under different experimental conditions, while CYP (cytochrome P450) and TUA (a tubulin) could act as reliable internal controls in different tissues. The reliable reference genes under treatment of hormone, heavy metal, salt, heat, cold, flooding and drought were EF1 alpha (elongation factor 1 alpha)/TIF5A (translation initiation factor 5A), UBQ10/18S (18S Ribosome RNA), UBQ10/CYP, TUB (beta-tubulin gene)/TIP41 (TIP41-like family protein), TIF5A/EF1 alpha, TUB/TIP41 and TIP41/PP2A (protein phosphatase 2A). Some common housekeeping genes, such as ACT (actin gene) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), exhibited unstable expression patterns. This was the first systematic study on the stability of internal reference genes of selection of reference genes in Carnation.

Automated summary

In this study, three computational statistical methods were used to evaluate the expression stability of 12 commonly-used reference genes in Dianthus caryophyllus. The results showed significant variations in the expression stability of candidate genes across different experimental conditions. TIP41 and UBQ10 were identified as relatively stable reference genes, while CYP and TUA could serve as reliable internal controls. Additionally, certain gene pairs were found to be suitable reference genes under specific treatments, such as hormone, heavy metal, salt, heat, cold, flooding, and drought.