Background: Quantification of gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) requires normalization to an endogenous reference gene termed housekeeping gene (HKG). Many of the commonly used HKGs are regulated and vary under experimental conditions and disease stages. Alcoholic liver disease (ALD) is associated with several different liver histological lesions that may modulate HKG expression. We investigated the variability of commonly used HGKs (18S, β-actin, glyceraldehyde-3-phosphate [GAPDH], and arginine/serine-rich splicing factor [SFRS4]) in the liver of patients with ALD.
Methods: Fifty consecutive patients at different stages of ALD underwent liver biopsy. The stability of HKG was assessed according to liver histological lesions.
Results: β-actin had the highest coefficient of dispersion (COD) (23.9). β-actin tended to decrease with steatosis and to increase with alcoholic hepatitis; β-actin also increased in patients with both alcoholic hepatitis and cirrhosis. GAPDH and SFRS4 COD were 2.8 and 2.1, respectively. GAPDH was decreased with steatosis and increased with alcoholic hepatitis and fibrosis. 18S had the lowest COD (1.4). Both 18S and SFRS4 levels were not significantly modified with respect to all alcohol-induced liver histological lesions.
Conclusions: In patients with ALD, the most constantly expressed HKGs are 18S and SFRS4. These genes are appropriate reference genes for normalization of RT-qPCR in the liver of patients with ALD. The use of other HKGs such as β-actin or GAPDH would lead to misinterpretation of the results.