Impact of culture medium on the interpretation of qRT-PCR data in HepG2 incubated with lactobacilli.

Recently, an increasing number of studies have investigated the mechanism of action of lactobacilli in the treatment of non-alcoholic fatty liver disease (NAFLD). Using four computational tools (NormFinder, geNorm, Delta Ct, and BestKeeper), six potential reference genes (RG) were analyzed in the human liver cell line HepG2 cultivated 24 hours in the presence of two strains of heat-killed lactobacilli, Limosilactobacillus reuteri E and Lactiplantibacillus plantarum KG4, respectively, in different cultivation media (DMEM high glucose (HG) or RPMI). The analysis revealed that the suitability of RG was similar between the two lactobacilli but quite different between the two media. The commonly used RGs, 18S rRNA and GAPDH were the most unstable in DMEM HG. Normalization of the mRNA expression of the target gene encoding sterol regulatory element-binding protein (SREBP-1c) to different RGs resulted in different expression profiles. This demonstrates that validation of candidate RGs under specific experimental conditions is crucial for the correct interpretation of qPCR data. In addition, the choice of media has a profound impact on the effect of lactobacilli on lipogenesis at the gene expression level, as shown by the transcription factor SREBP-1c.