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Equine sarcoids, the most common skin tumours in horses, are induced by bovine papillomavirus (BPV). Their clinical appearance varies from small stable patches to aggressively growing masses. Differences in BPV load and mRNA expression and Ki67 and p53 immunostaining among four clinical types (fibroblastic, occult, nodular and verrucous sarcoids) were evaluated to test the hypothesis that the clinical behaviour of equine sarcoids correlates with BPV activity. Viral load and expression of the BPV E2, E5, E6 and E7 genes were determined using quantitative real-time PCR. The proliferative fraction (PF) of the tumours was determined by Ki67 immunostaining and expression of p53 was analysed by immunohistochemistry. Nodular sarcoids showed a significantly higher viral load than the other types. A significant overall difference among the four types was observed for E2, E5, E6 and E7 mRNA expression. Nodular sarcoids showed the highest expression level for each BPV gene examined, followed by verrucous, fibroblastic and occult tumours. Viral DNA and mRNA outcomes correlated with each other, indicating a similar transcription pattern in each type of sarcoid. The PF was significantly higher in the superficial layers of verrucous and fibroblastic sarcoids compared with occult and nodular types. No significant difference was observed for the PF in the deep layers and for p53 expression. These results clearly demonstrate the omnipresence and active transcription of BPV in equine sarcoids. However, the hypothesis that the clinical behaviour of an equine sarcoid can be explained on the basis of differences in BPV activity could not be demonstrated. INTRODUCTIONEquine sarcoid is the most common skin tumour in horses. The clinical aspect can vary remarkably, ranging from small, stable patches to aggressively growing tumours up to more than 10 cm in diameter. A sarcoid can appear as a single tumour, but horses often show multiple lesions. Although equine sarcoids do not metastasize, they recur frequently if not treated properly. Different clinical types have been described (Pascoe & Knottenbelt, 1999). The fibroblastic type, with a typically ulcerated epidermis, usually presents an aggressive clinical behaviour (Fig. 1a). Occult types are flat, alopecic patches, sometimes with hyperkeratosis or small nodules on their surface (Fig. 1b). These tumours will often not evolve, even over several years. Nodular sarcoids are round masses with an intact epithelium (Fig. 1c). The overlying skin can be loose from the stromal tissue or (partially) attached to the fibroblastic portion of the tumour. Verrucous sarcoids have a typically warty appearance (Fig. 1d). Nodular and verrucous sarcoids display a clinical behaviour in between the fibroblastic and the occult sarcoid, mostly with moderate growth. Mixed sarcoids, composed of two or more of the described types, are also commonly observed. Occult sarcoids, and even nodular and verrucous sarcoids, can remain stable for many years and then suddenly, without any apparent reason, change dramatica...

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Background: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on realtime PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses.