| Summary: | <p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Six genes, beta-2-microglobulin (<it>B2M</it>), glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), hydroxymethyl-bilane synthase (<it>HMBS</it>), hypoxanthine phosphoribosyl-transferase 1 (<it>HPRT1</it>), succinate dehydrogenase complex, subunit A (<it>SDHA</it>) and ubiquitin C (<it>UBC</it>), 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 <it>geNorm </it>and <it>NormFinder </it>software.</p> <p>Results</p> <p><it>HMBS </it>and <it>GAPDH </it>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. <it>HMBS, GAPDH </it>and <it>UBC </it>were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of <it>HMBS, B2M</it>, <it>SDHA </it>and <it>GAPDH </it>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.</p> <p>Conclusion</p> <p>Of six genes studied, <it>HMBS </it>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.</p>
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