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

We evaluated 6 commonly used housekeeping genes' expression levels in 108 HBV-related HCCs' matched tumor and non-tomor 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.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.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.With the application of quantitative real-time polymerase chain reaction (qPCR) in the high throughput and accurate expression profiling of selected genes, gene expression analysis is increasingly significant in many fields of biological research [1-3]. Nowadays, housekeeping genes (HKGs) are routinely-used as references in qPCR to normalize experimental data, such as differences in RNA quantity and quality, the overall transcriptional activity and differences in the cDNA synthesis [4], because, theoretically, HKGs are supposed to exhibit consistent, non-regulated, stable expression among different space-time and different tissues, even intervention models [5,6].However, cancer development is a very complex stepwise process involving altered cell function