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Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT-qPCR data. The key aim of this method is to identify an applicable internal reference gene. However, there are currently no data concerning the expression of reference genes for gene analysis in human tongue carcinoma cell lines and tissues. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in human tongue carcinoma. Tca-8113 and CAL-27 cell lines and a total of 8 tongue carcinoma tissue samples were investigated. The gene expression stability and the applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. The results from the three software programs were demonstrated to be variable following comparison. The recommended combinations were 5'-aminolevulinate synthase 1 + glucuronidase β + ribosomal protein L29 (RPL29) for the cell line + tissue group, β-2-microglobulin + RPL29 for the cell line group and peptidylprolyl isomerase A + hydroxymethylbilane synthase + RPL29 for the tissue group. These recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on human tongue carcinoma.