Dataset Information

Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses.

ABSTRACT: BACKGROUND: Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different clinical outcomes. METHODS: We evaluated 6 commonly used housekeeping genes' expression levels in 108 HBV-related HCCs' matched tumor and non-tumor tissue samples with different clinical outcomes and 26 normal liver specimens by real-time PCR. The expression stability of the 6 genes was compared using the software programs geNorm and NormFinder. To show the impact of reference genes on data analysis, we took PGK1 as a target gene normalized by each reference gene, and performed one-way ANOVA and the equivalence test. RESULTS: With the geNorm and NormFinder software programs, analysis of TBP and HPRT1 showed the best stability in all tissue samples, while 18s and ACTB were less stable. When 18s or ACTB was used for normalization, no significant difference of PGK1 expression (p > 0.05) was found among HCC tissues with and without metastasis, and normal liver specimens; however, dramatically differences (p < 0.001) were observed when either TBP or the combination of TBP and HPRT1 were selected as reference genes. CONCLUSION: TBP and HPRT1 are the most reliable reference genes for q-PCR normalization in HBV-related HCC specimens. However, the well-used ACTB and 18S are not suitable, which actually lead to the misinterpretation of the results in gene expression analysis.

SUBMITTER: Fu LY

PROVIDER: S-EPMC2644316 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses.

Fu Li-Yun LY Jia Hu-Liang HL Dong Qiong-Zhu QZ Wu Jin-Cai JC Zhao Yue Y Zhou Hai-Jun HJ Ren Ning N Ye Qin-Hai QH Qin Lun-Xiu LX

BMC cancer 20090206

<h4>Background</h4>Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepato ...[more]

PMID: 19200351

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Project description:BackgroundQuantitative real-time PCR (qPCR) is a widely used technique for gene expression analysis. Its reliability is highly dependent upon selection of the appropriate reference genes for accurate gene expression normalization. In this study, we investigated the expression stability of 10 commonly used reference genes in a mouse myocardial infarction model.Methods & resultsThe expression stability of the 10 reference genes (Actb, B2m, Eef1a1, Gapdh, Hprt, Polr2a, Ppia, Rpl13a, Tbp, Tpt1) was analyzed using the geNorm software. Overall, the combination of Hprt, Rpl13a and Tpt1 was the most stable reference gene set in our experiments. Gapdh, Polr2a and Actb consistently showed the highest gene expression variability and the expression levels of Gapdh, Polr2a, Actb, B2m and Eef1a1 were found to be selectively up- or downregulated after myocardial infarction. We normalized the expression of Nppb and Vcam1, using different reference gene strategies and demonstrated that their induction after myocardial infarction was most clearly revealed with the optimal reference gene combination. However, the use of suboptimal reference gene combinations resulted in detrimental effects on gene expression levels and variability with a gradual loss of the expression differences and a significant reduction in statistical power.ConclusionsHprt, Rpl13a and Tpt1 are a set of stably expressed reference genes for accurate gene expression normalization in myocardial infarction studies in mice. We found that Gapdh, Polr2a and Actb display high expression variability in mouse myocardial infarction tissues and that loss of statistical power and increase in sample size are the evident consequences of choosing suboptimal combinations of reference genes. We furthermore caution against the use of Gapdh, Polr2a, Actb, B2m and Eef1a1 for gene expression normalization in myocardial infarction studies because of selective up- or downregulation after myocardial infarction, which could potentially lead to biased study outcomes.

Project description: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.

Dataset Information

Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses.

Publications

Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses.

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