Figure 1.
Liver damage assessment in rat models of acute hepatotoxicity.
Plasma alanine aminotransferase (ALT) levels and histological examination of the livers from rats after 24 h of intraperitoneal administration of acetaminophen (panel A), carbon tetrachloride (panel B), D-galactosamine (panel C) and thioacetamide (panel D) are shown. The change in the plasma ALT levels in response to increasing doses of each hepatotoxin was tested by a one-way ANOVA followed by the Newman–Keuls test for multiple comparisons. An asterisk (*) indicates a significant difference (p<0.05). Representative histological microphotographs of hematoxylin and eosin stained-liver sections after the administration of highest doses of the hepatotoxins and their respective vehicles (big and small image of each panel, respectively) are shown. The rats were intraperitoneally administered with: acetaminophen (1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight).
Table 1.
Descriptions of selected candidate reference genes and genes of interest.
Table 2.
Descriptions of gene-specific real-time PCR assays.
Figure 2.
Boxplot of RT-qPCR quantification cycles values of candidate reference genes.
Boxplot of quantification cycles (Cq) values for each reference gene for microRNA and mRNA normalization in all liver samples assessed (n = 35) which belong to rats treated with each one of the four hepatotoxins studied and their respective control animals administered with vehicle. A line across the box depicts the median. The box indicates the 25% and 75% percentiles. Whiskers represent the maximum and minimum values, circles represent outliers. The livers were evaluated 24 h after rats were intraperitoneally administered with: acetaminophen (1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight).
Figure 3.
Effect of rat exposure to hepatotoxins on liver expressions of candidate reference genes.
The expression of candidate reference genes for microRNA and mRNA normalization in the liver of rats treated with the hepatotoxins and their respective vehicles are shown. The livers were evaluated 24 h after rats were intraperitoneally administered with: acetaminophen (AA, 1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (CT, 1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (GA, 0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (TA, 150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight). The bars represent the means of the relative quantity of transcript ± SD of 5 animals treated with hepatotoxins or their respective vehicle. An asterisk (*) indicates a significant expression difference with p<0.05 using Student's t-test of the log-transformed data between the control and treated groups.
Table 3.
Ranking of candidate reference genes according to their stability value.
Figure 4.
Study of the normalization efficiency of candidate reference genes.
The average normalization efficiency index (NEI) of the four treatments for each microRNA or mRNA normalization method and their corresponding standard deviation are shown in up- and down-regulation situations. For each treatment with a specific hepatotoxin, we defined the NEI value as the minimum fold up- or down-regulation of the simulated expression of a hypothetical gene needed to observe a significant difference (t test, p = 0.05, n = 5) using different methods of normalization. The liver expression value of one or combination of reference genes (RGs) was used to normalize the expression of the hypothetical gene. The liver expression of RGs were assessed 24 h after rats were intraperitoneally administered with: acetaminophen (AA, 1.2 g/kg body weight) or its vehicle (1% carboxymethyl cellulose, 10 ml/kg body weight); carbon tetrachloride (CT, 1 ml/kg body weight) or its vehicle (corn oil, 4.4 ml/kg body weight), D-galactosamine (GA, 0.9 g/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight); thioacetamide (TA, 150 mg/kg body weight) or its vehicle (saline solution, 6 ml/kg body weight).
Figure 5.
Workflow for reference gene selection.
All the steps (S+number) followed for reference gene selection in this report are shown. The strategy (S) applied and the tools (T) used in each step are mentioned in each corresponding panel.