Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity

Area of Science:

• Toxicology
• Molecular Biology
• Genetics

Background:

• Hepatotoxicity involves significant liver gene expression changes due to xenobiotic exposure, necessitating understanding of underlying mechanisms for diagnosis and treatment.
• MicroRNAs (miRNAs) are critical gene expression regulators, and their quantification via RT-qPCR requires stable endogenous reference genes for accurate results.
• Candidate reference gene stability is highly variable, making the identification of optimal genes essential for reliable gene expression analysis in specific experimental contexts.

Purpose of the Study:

• To identify and validate optimal reference gene combinations for normalizing microRNA (miRNA) and messenger RNA (mRNA) quantitative PCR (qPCR) data in experimental models of acute hepatotoxicity.
• To assess the expression stability of commonly used and novel candidate reference genes under hepatotoxic conditions induced by acetaminophen, carbon tetrachloride, D-galactosamine, and thioacetamide.
• To ensure accurate gene expression profiling in hepatotoxicity research by establishing reliable normalization strategies compliant with MIQE guidelines.

Main Methods:

• Rats were administered four common hepatotoxins to induce acute liver injury.
• Liver tissue samples were analyzed using RT-qPCR to determine the expression levels of candidate reference genes for both miRNA and mRNA normalization.
• Expression stability of candidate genes was evaluated using geNorm, NormFinder, and BestKeeper software, followed by validation of optimal combinations.

Main Results:

• Traditional reference genes (U6, Beta Actin, Glyceraldehyde-3P-dehydrogenase) exhibited unstable expression in response to hepatotoxins, rendering them unsuitable for normalization.
• Novel reference gene combinations, specifically miR-16/5S rRNA for miRNA and B2M/18S rRNA for mRNA, demonstrated high stability across different hepatotoxic models.
• The validated reference gene sets provide accurate normalization for miRNA and mRNA quantification in experimental acute hepatotoxicity.

Conclusions:

• Standard reference genes are unreliable for miRNA and mRNA normalization in experimental hepatotoxicity models.
• The identified optimal reference gene combinations (miR-16/5S rRNA and B2M/18S rRNA) are validated for accurate gene expression analysis in acute rat liver injury.
• These findings provide a robust normalization strategy crucial for advancing the understanding and clinical management of drug-induced liver injury.