Improvement of Gene Expression Studies in the DimethylnitrosamineInduced Liver Fibrosis Model in the Rat Using Selected Reference Genesfor Quantitative Real Time-PCR Analysis

Background: Liver fibrosis is a reaction to chronic liver injury characterized by excessive accumulation of collagen. Due to their importance as biomarkers, the changes in gene expression in the liver during the development of fibrosis and its subsequent outcomes of cirrhosis, neoplasia or resolution are intensely studied. Quantitative realtime PCR (qPCR) with its ability to detect and measure minute amounts of nucleic acids have been increasingly used in these studies. In qPCR, the quantitation of mRNA is relative and the accuracy of results dependent on the reference genes used for standardization. However, many genes studied are normalized against single reference genes, usually housekeeping genes, without adequate justification. Methods: For the dimethylnitrosamine (DMN) induced liver fibrosis rat model, we tested 8 commonly used candidate genes (Actb, Alb, Sdha, B2m, Rn18s, Hprt1, Ppia and Gapdh) to determine their suitability as reference genes. qPCR results were analysed using four commonly used programs; NormFinder, GeNorm, Comparative ΔCt methods and BestKeeper. Result: It was determined that Gapdh and B2m were the most stable genes in normal liver. However, in DMN treated livers, Gapdh and Ppia were the most stably expressed reference genes. We validated these reference genes by using them to normalize the expression of four genes; Tgfb 1, Col1a1, Col3a1 and Tnf known to be highly expressed in liver fibrosis. Conclusion: Gapdh and Ppia are the most suitable reference genes for the normalization of qPCR data in gene expression studies of the liver in the DMN induced liver fibrosis model in the rat. We advise against the use of Actb in this experimental setting because of its low expression stability.