Abstract
BackgroundReference 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.MethodsSix 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.ResultsHMBS 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.ConclusionOf 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.
Highlights
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
Six putative reference genes from different abundance and functional classes were selected for evaluation: B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase flavoprotein subunit A (SDHA), and ubiquitin C (UBC) (Table 1)
RNA samples were subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) inhibitor detection for the mining of the relationship between optical density (OD) 260/230 ratio and quantitative reverse transcription (qRT)-PCR inhibition
Summary
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). Real-time quantitative reverse transcription (qRT)polymerase chain reaction (PCR) is a rapid, sensitive and reliable method for gene expression studies. It is inherently an indirect method of measurement, and variabilities exist in the various steps of the qRT-PCR which may lead to severe misinterpretation of the results. Various strategies have been explored in an attempt to normalize these variations, and it is generally accepted that gene-expression levels should be normalized by carefully selected and stably expressed reference genes [2,3,4]. It is critical to perform preliminary evaluation studies, aimed at identifying the most stably expressed reference genes in individual tissues and distinct circumstances for each single experiment
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