Selection of reference genes for quantitative real-time reverse transcription-polymerase chain reaction in concanavalin A-induced hepatitis model

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Abstract

Quantitative real-time reverse transcription-polymerase chain reaction (Q-PCR) has become an indispensable technique for accurate determination of gene expression in various samples. In mice, intravenous injection of concanavalin A (ConA) leads to acute hepatitis and liver injury. Functional studies based on this model have provided insights for understanding the mechanisms of liver injury. However, no data have been reported to validate reference genes during the progression of ConA-induced hepatitis (CIH). In this study, IκBα and C/EBPβ messenger RNA (mRNA) levels were examined using Q-PCR with ACTB as the reference gene after ConA injection. However, we got inconsistent results with previous reports determining IκBα and C/EBPβ mRNA expression levels. The results indicate the necessity for stability analysis of candidate reference genes in the CIH model. geNorm, NormFinder, and BestKeeper software analysis indicates that ACTB is the most unstable gene during CIH progression among the 10 reference genes tested, whereas RPLP0 or HPRT1 is the most stable one. This study demonstrates that some of the commonly used reference genes are inadequate for normalization of Q-PCR data due to their expression instability. Furthermore, this study validates HPRT1 and RPLP0 as appropriate reference genes for Q-PCR analysis in the CIH model.

Section snippets

Animal treatment

C57BL/6 male mice, 8–10 weeks of age, were purchased from Shanghai Laboratory Animal Company (SLAC, Shanghai, China). Mice were housed in the animal facilities of the Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Jiao-Tong University School of Medicine, under pathogen-free conditions according to the institutional animal care and use committee guidelines. Mice were fed ad libitum a standard laboratory chow diet provided by SLAC.

CIH model

Mice (4 or 5 per group) were injected via the

ConA treatment induces hepatitis and hepatocellular apoptosis

Hepatic changes were examined histologically and biochemically at different time points after ConA injection. Consistent with a previous report [23], serious necrotic phenotypes were observed in livers at 8 and 24 h after ConA injection, whereas the necrotic phenotype nearly disappeared at 72 h (Fig. 1A). Simultaneously, based on the genomic DNA fragmentation analysis [16], hepatocytes demonstrated obvious apoptosis at 8 h as compared with the control group, and even more serious at 24 h,

Discussion

Hepatitis is a potentially life-threatening liver disease. Mouse hepatitis models provide valuable insights in attenuating and curing the disease. CIH in mouse is a model developed during recent years and is used with increasing interest to investigators of liver disease [32]. Gene expression profiles at mRNA and protein levels during hepatitis progression are extremely valuable for functional analysis. Q-PCR analysis provides great convenience and accuracy in determination of target gene mRNA

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (2006AA02A409) and the National Natural Science Foundation of China (30973571).

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