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Eleven reference genes (18s ribosomal ribonucleic acid [RNA], 28s ribosomal RNA, ubiquitin, beta-actin, glycerine aldehyde dehydrogenase, ATP-synthase subunit 5B, hydroxymethyl-bilane synthase, hypoxanthine-phosphoribosyl transferase, ribosomal protein L32, tryptophan 5-monooxygenase activation protein (zeta polypeptide), and TATA-Box binding protein) were analyzed in use as references for gene expression profiling experiments using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine mammary tumors. The transcription level of the candidates was measured in 22 histologically characterized excised tumor specimens from mammary gland tissue and 22 samples of non-neoplastic mammary tissue samples from the same individuals. Results were used to rank candidate reference genes using the GeNorm tool. It was determined that in samples of canine mammary gland tissue, a combination of hypoxanthine-phosphoribosyl transferase, ATP-synthase subunit 5B, ribosomal protein L32 and ubiquitin yields stable reference gene expression levels, whereas the use of glycerin aldehyde dehydrogenase or ribosomal RNA is unsuitable for normalization of qRT-PCR results in this tissue type.