Abstract
The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.
Highlights
Qualitative and quantitative analyses, of gene and protein expression, rely on the presence of reference genes and proteins that are expressed at constant levels in the analyzed samples
The expression levels of β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin were shown to increase in some tumors [1, 2], while other conditions such as Alzheimer’s disease, alcoholic hepatitis, cirrhosis, and schizophrenia have been implicated in altering the expression of endogenous reference genes and/or proteins [3, 4]
When comparing the ob/ob group to the high-fat diet (HFD) group, there was an observable reduction in the amounts of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%)
Summary
Qualitative and quantitative analyses, of gene and protein expression, rely on the presence of reference genes and proteins that are expressed at constant levels in the analyzed samples. Inappropriate endogenous controls can compromise the accuracy and reliability of the results. This is because these genes and proteins are commonly used to normalize data and correct experimental errors, allowing for direct comparisons of gene and/or protein expression [1]. Previous studies have shown that the expression of some internal controls varies, depending on the experimental conditions. The expression levels of β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin were shown to increase in some tumors [1, 2], while other conditions such as Alzheimer’s disease, alcoholic hepatitis, cirrhosis, and schizophrenia have been implicated in altering the expression of endogenous reference genes and/or proteins [3, 4]. Nonalcoholic fatty liver disease (NAFLD) has been shown to alter the constitutive expression of reference genes used to normalize gene expression levels [5]
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