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<p>Abstract</p> <p>Background</p> <p>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 real-time PCR in horses, normalisation occurred against only one reference gene, usually <it>GAPDH </it>or <it>ACTB</it>, 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.</p> <p>Results</p> <p>In the present study the gene transcription levels of 6 commonly used reference genes (<it>ACTB, B2M, HPRT1, UBB, TUBA1 </it>and <it>RPL32</it>) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, <it>TUBA1, ACTB </it>and <it>UBB </it>were found to be most stable in normal skin and <it>B2M, ACTB </it>and <it>UBB </it>in equine sarcoids.</p> <p>Conclusion</p> <p>Based on these results, <it>TUBA1, ACTB </it>and <it>UBB</it>, respectively <it>B2M, ACTB </it>and <it>UBB </it>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 <it>UBB, ACTB </it>and <it>B2M </it>can be recommended as a reliable and accurate normalisation factor.</p>