Abstract
BackgroundReal-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 real-time 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.ResultsIn the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids.ConclusionBased on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.
Highlights
Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions
It has been demonstrated that the expression levels of these genes may vary considerably in different tissues, different cell types and different disease stages, in clinical samples associated with malignant disease [3,4]
Transcription profiling of the candidate genes cDNA was synthesised from DNA-free RNA isolated from 8 normal equine skin and 8 equine sarcoid samples
Summary
Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. 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, in clinical samples associated with malignant disease. Some housekeeping genes may have a specific function essential for the tumour metabolism and be up or down regulated [8] Because of these findings, Vandesompele et al [9] proposed to identify a set of stable housekeeping genes in the tissue of interest and use them as internal reference genes for accurate normalisation
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