To investigate the influence of the animal health status on the stability of the candidate reference genes, the analyses were performed in whole blood of control sheep and of sheep showing disease symptoms after clinical evaluation. Moreover, disease-stressed animals were sampled and analysed twice in order to monitor gene stability in disease-stressed sheep not only at different time points, but also under heat stress conditions, which, in association to disease status, really represent an extreme situation (see the Experimental Section for details on animal selection and sampling procedure). In all sheep groups, the results obtained with geNorm and Normfinder were consistent although not identical, as similarly reported elsewhere [19] [20] [21] [22] . SDHA and YWHAZ can be considered the most stably expressed genes in ovine whole blood ranking at the top positions in the control and disease-stressed sheep, both when they were analysed separately and when they were combined. SDHA and YWHAZ stability appears to be reliable as it was affected neither by disease status alone nor in association with heat stress. Moreover, geNorm indicated SDHA/YWHAZ as the best reference gene combination in the control and disease-stressed sheep joined datasets, a situation likely to fit most experimental contexts involving case and control animals; however, the SDHA/YWHAZ combination would be suitable for normalization of gene expression data also in studies carried out under physiological conditions, as these two genes demonstrated high stability in the control sheep group as well. Although data in sheep are still limited, SDHA appears to have good stability in this species as it was included in the reference genes required for reliable normalisation in several tissues (cerebrum, spleen, mesenteric lymph node and ileum). Similarly, YWHAZ was included in the optimal panel of reference genes to be used in the cerebellum, obex and ileum [5] . HPRT expression stability was evaluated only in the lung and pulmonary artery of brainstem death and control sheep, but it performed poorly as reference in both tissues on separate and combined analyses [7] . Actually, HPRT ranked as the third most stable gene of the control group in sheep whole blood, but its stability strongly decreased under disease conditions, emphasizing that proper validation of reference genes in a cell type or tissue of interest and under different experimental settings is mandatory before reporting qRT-PCR results. B2M showed stable expression in one study on human leukocytes from 13 healthy donors [1] . B2M also had stable expression in a large study in which 526 human whole blood samples represented healthy individuals and six disease groups [23] . In sheep, however, B2M is outperformed by other genes and demonstrates suboptimal suitability as reference gene in whole blood. In humans, GYPC expression is considerably higher in erythroid lineage cells than in non-erythroid cells [24] . GYPC was therefore used by Brown et al. (2007) to normalize qRT-PCR analyses of erythroid markers in sheep whole blood [11] . In our study, however, both geNorm and NormFinder classified GYPC in the bottom half of the stability ranking under both control and disease conditions, showing that ubiquitously expressed genes would provide a more relevant comparison for measuring erythroid gene expression. In control sheep, GAPDH resulted in being the gene with the highest degree of individual variation in expression level. This finding was not surprising, as GAPDH can be regulated under a large number of physiological states and is generally not considered a good reference gene [25, 26] . Nevertheless, GAPDH ranked as the second most stable gene in the disease-stressed and the combined sheep groups. Taylor et al. (2008) found that the levels of GAPDH transcription were the most stable of the genes tested in ovine peripheral blood mononuclear cells during infection with Mycobacterium avium subsp. paratuberculosis [9] . This finding is consistent with our results indicating stability of GAPDH in whole blood under disease condition. However, it must be taken into account that Taylor's results could also be attributable to a characteristic of the specific cell type or to the fact that the study based its observations on analysis of the standard deviation of C t values and not on specifically designed algorithms.