Selection of Reference Genes for Real-time Polymerase Chain Reaction in Canine Oral Tumor and Cancer

Abstract

Tumors in oral cavity either benign or malignant are often found in dogs. Despite the increasing number of studies of gene expression associated with canine oral cavity diseases, reference gene selection for normalizing target genes has not been performed. The objective of the present study was to identify potential reference genes in canine oral tumors, including epulis, ameloblastoma, oral melanoma, oral squamous cell carcinoma, etc. Suitability of 6 candidate reference genes, beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), ribosomal subunit L13a (RPL13a), ribosomal protein S5 (RPS5) and ribosomal protein S19 (RPS19), was evaluated with 5 algorithms, geNorm, Normfinder, BestKeeper, the comparative ΔCt method and RefFinder. Except Bestkeeper, most algorithms showed that a cohort of RPS5, RPS19 and ACTB was most suitable to normalize target genes. Since some algorithms did not show the same ranks of reference genes, it is necessary to normalize target genes against more than 1 reference gene with more than 1 algorithm. In conclusion, this work suggests that ACTB in combination with RPS19 and RPS5 are validated to be used as reference genes for expression study in canine oral tumors. These reference genes will increase the reliability of any qRT-PCR gene expression analysis of canine oral tumor and cancer in several aspects such as clinical diagnosis, prognosis and drug testing.