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

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Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

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.

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Abbreviations

RT-PCR:

Reverse transcriptase polymerase chain reaction

HKG:

Housekeeping gene

HCV:

Hepatitis C virus

HCC:

Hepatocellular carcinoma

HBV:

Hepatitis B virus

GDC:

Genomic DNA Control

PPC:

Replicate positive PCR control

NTC:

No template control

Ct:

Threshold cycle

CV:

Coefficient of variation

TNM:

Tumor-node-metastasis

AFP:

Alpha-fetoprotein

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Acknowledgments

This study was supported by grants from Key Project of the Chinese Ministry of Education (No. 107039) and the Foundation of China National “211” Project for Higher Education. We thank Kangchen Bio-technology Company (Shanghai, People’s Republic of China) for technical support.

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Authors and Affiliations

  1. Key Laboratory for Carcinogenesis and Cancer Invasion, The Chinese Ministry of Education, Liver Cancer Institute, Zhong Shan Hospital and Shanghai Medical School, Fudan University, 136 Yi Xue Yuan Road, Shanghai, 200032, People’s Republic of China

    Qiang Gao, Xiao-Ying Wang, Jia Fan, Shuang-Jian Qiu, Jian Zhou, Ying-Hong Shi, Yong-Sheng Xiao, Yang Xu, Xiao-Wu Huang & Jian Sun

Authors
  1. Qiang Gao
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  2. Xiao-Ying Wang
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  3. Jia Fan
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  4. Shuang-Jian Qiu
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  5. Jian Zhou
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  6. Ying-Hong Shi
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  7. Yong-Sheng Xiao
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  8. Yang Xu
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  9. Xiao-Wu Huang
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  10. Jian Sun
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Corresponding author

Correspondence to Jia Fan.

Additional information

Qiang Gao and Xiao-Ying Wang contributed equally to this work.

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Gao, Q., Wang, XY., Fan, J. et al. Selection of reference genes for real-time PCR in human hepatocellular carcinoma tissues. J Cancer Res Clin Oncol 134, 979–986 (2008). https://doi.org/10.1007/s00432-008-0369-3

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  • DOI: https://doi.org/10.1007/s00432-008-0369-3

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