Evaluation of reference genes for qPCR in human liver and kidney tissue from individuals with obesity

Given the obesity epidemic and the prevalence of comorbidities, there is an ongoing need to understand the health consequences of this disease state better. Understanding gene expression signals will facilitate the identification of mechanisms of kidney and liver dysfunction/disease often present in individuals with obesity. qPCR is the standard method for studying changes in relative gene expression. Reference genes (RGs) are obligatory for accurately normalizing mRNA transcript levels across samples. Despite the prevalence of qPCR, the reliability of the data is often compromised because RGs are still used without validation or have proven to be unstable in different tissues and various diseases. In this study, we validated seven reference genes (ACTB, B2M, RPLP0, HPRT1, GAPDH, 18S rRNA, and PPIA) using human liver tissue from 15 lean individuals and 17 individuals with a BMI ≥ 25 and human kidney tissue from 13 lean individuals and 15 individuals with a BMI ≥ 25. Cross-validation of expression stability was performed using the RefFinder platform with four algorithms: NormFinder, BestKeeper, geNorm, and the comparative ΔCt method. In obesity-related studies, the most suitable reference genes in gene expression studies are RPLP0 and HPRT1 in human kidney tissue and RPLP0 and GAPDH in the liver.