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Background  Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.    Methods  Six genes, beta-2-microglobulin ( B2M ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), hydroxymethyl-bilane synthase ( HMBS ), hypoxanthine phosphoribosyl-transferase 1 ( HPRT1 ), succinate dehydrogenase complex, subunit A ( SDHA ) and ubiquitin C ( UBC ), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both  geNorm  and  NormFinder  software.    Results   HMBS  and  GAPDH  were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC.  HMBS, GAPDH  and  UBC  were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of  HMBS, B2M ,  SDHA  and  GAPDH  was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.    Conclusion  Of six genes studied,  HMBS  was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR