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Background  Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so 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  We used three different algorithms (BestKeeper, geNorm and NormFinder) 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-1α, 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-α  and  Tubulin . We identified  PP2A ,  SAND  and  UBC  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 reference genes, so that future research should also consider different genotypes and ploidy levels.

Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)