Selection of reference genes for real-time PCR in human hepatocellular carcinoma tissues

Aims To investigate the most suitable housekeeping genes for quantifying a change in mRNA expression levels due to hepatitis virus B related hepatocellular carcinoma (HCC). Methods Expression of mRNA encoding ACTB, GAPDH, B2M, HPRT and TBP was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in matched malignant and non-malignant tissues obtained from 65 non-treated HCCs. The software programs geNorm and NormFinder were used to ascertain the most suitable reference gene combination. Results All candidate genes showed significantly different expression between malignant and non-malignant samples. GAPDH and ACTB, genes most frequently used for normalization, were heavily regulated during HCC carcinogenesis and progression. B2M expression levels varied with hepatitis infection status. The combination of HPRT and TBP expression levels were the most stable, regardless of differences in tumor stage and cirrhotic and malignancy status. Conclusions It is necessary to select reference genes based on tissue and disease specific expression profile and to further identify novel reference genes with greater expression stability for use in HBV related HCC gene expression studies.