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Identifying superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses
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To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, also called reference genes. However, recent studies show there is no universal reference gene for all biological questions.
Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies.
Results: Three different algorithms (BestKeeper, geNorm and NormFinder) were used to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes"(Actin, EF-1alpha, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC).
The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes.
In general, Tubulin showed the most variable expression and should be avoided as a reference gene.
Conclusions: Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-alpha and Tubulin.
PP2A, SAND and UBC were identified as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus Rosa, including differences in ploidy levels, might also influence expression stability of referencegenes, so that future research should also consider different genotypes and ploidy levels.
