Identification of stably expressed Internal Control Genes (ICGs) for normalization of expression data in liver of C57BL/6 mice injected with beta casomorphins

In recent years, beta-casomorphin peptides (BCM7/BCM9) derived from the digestion of cow milk have drawn a lot of attention world over because of their proposed impact on human health. In order to evaluate the transcriptional modulation of target genes through RT-qPCR in response to these peptides, availability of appropriate reference or internal control genes (ICGs) will be the key. The present study was planned to identify a panel of stable ICGs in the liver tissue of C57BL/6 mice injected with BCM7/BCM9 cow milk peptides for 3 weeks. A total of ten candidate genes were evaluated as potential ICGs by assessing their expression stability using software suites; geNorm, NormFinder and BestKeeper. The suitability of the identified ICGs was validated by assessing the relative expression levels of target genes, HP and Cu/Zn SOD. Based on geNorm, PPIA and SDHA gene pair was identified to be most stably expressed in liver tissue during the animal trials. Similarly, NormFinder analysis also identified PPIA as the most stable gene. BestKeeper analysis showed crossing point SD value for all the genes in the acceptable range that is closer to 1. Overall, the study identified a panel of stable ICGs for reliable normalization of target genes expression data in mice liver tissues during BCM7/9 peptides trial.

Automated summary

In recent years, there has been a lot of interest in beta-casomorphin peptides (BCM7/BCM9) derived from cow milk digestion and their potential impact on human health. This study aimed to identify stable internal control genes (ICGs) for the reliable evaluation of target gene expression in the liver tissue of mice injected with BCM7/BCM9 peptides. Ten candidate genes were evaluated for their expression stability using geNorm, NormFinder, and BestKeeper software. PPIA and SDHA were identified as the most stably expressed genes in the liver tissue, based on geNorm and NormFinder analysis, while BestKeeper analysis showed acceptable crossing point SD values for all genes. Overall, this study identified a panel of stable ICGs for the normalization of target gene expression data in mice liver tissues during BCM7/9 peptides trial.