Exploring transcriptomic databases to identify and experimentally validate tissue-specific consensus reference gene for gene expression normalization in BALB/c mice acutely exposed to 2,3,7,8-Tetrachlorodibenzo- p-dioxin

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic compound affecting organs like the liver, kidney, lung, and reproductive systems in mammals. This study outlines a strategy for choosing appropriate HKGs for tissue-specific gene expression analysis in TCDD toxicity, including four steps: i) identifying candidate HKGs from literature and databases; ii) defining primers from literature or designing new ones; iii) validating primer efficiency and specificity; iv) experimentally assessing candidate HKGs' stability in various tissues of TCDD-exposed mice. Based on this strategy, a total of 40 potential HKGs was selected, further filtered based on their database sources and ranked according to their frequency of use or expression stability. Ultimately, we identified a final set of 15 HKGs (Rps18, Calr, Polr2b, Brms1l, P4hb, Esd, Hdgf, Gapdh, Mlec, Tbp, Rn18s, Sdha, B2m, Actr3 and Actb) with typical efficiencies for further evaluation. Then, the stability of the selected HKGs was determined in the liver, kidney, lung, ovary and testis of TCDD-exposed mouse compared to the control group using the [log (2^ΔCt)] and statistically analyzed using Pearson correlation coefficient (r) by BestKeeper algorithm. Our data analysis revealed that Actb, Rps18, and Polr2b were the most stable HKGs for normalizing gene expression in the liver, while Sdha, Actb, and Gapdh were suitable for kidney tissue. In the lung, Tbp, Sdha, and Rps18 showed stability, while Tbp, B2m, and Actb were most stable in ovary. Lastly, Actb, B2m, and Tbp were accurately stable in the testis of TCDD-exposed mice. Our study identifies stable HKGs, improving TCDD toxicity research accuracy and reliability.

Keywords: Dioxin; Gene expression; Housekeeping genes; Mouse BALB/C; RT-qPCR.

Graphical abstract

Fig. 1

A. A schematic representation of workflow. The workflow was structured into three main steps: first, the primary selection of housekeeping genes (HKGs) from transcriptomic databases (PUBMED literature and HRT Atlas) (indicated by blue boxes); second, the validation of primers and amplification conditions for the selected HKGs (indicated by green boxes); and finally, the assessment of the stability of the chosen 15 HKGs in the liver, kidney, lung, ovary, and testis of mice exposed to TCDD. B. Initially, a total of 40 HKGs were identified as potential candidates for normalizing gene expression; 23 from PUBMED and 17 from the HRT Atlas database. Among the 23 HKGs sourced from toxicological studies in mice, their frequency of use was ranked, represented as a percentage. Subsequently, the top-ranked 15 HKGs were selected for further assessment. As for the 17 HKGs retrieved from the HRT Atlas databases, they were evaluated for expression stability based on mean RPKM (Reads Per Kilobase Million), standard deviation of log2 RPKM, and MFC (Median Fold Change) value. The top 5 HKGs were then designated as potential candidates for further evaluation.

Fig. 2

Coefficient of intra-assay and inter-assay variation for the amplification of selected HKGs. A. Mean Ct values for six technical replicates, indicated by dots, for each candidate reference gene obtained from a single run (intra-assay variation). Horizontal lines depict the mean value for each group. B. Mean Ct values for six technical replicates for each candidate reference gene obtained from three different runs on separate days (inter-assay variation).

Fig. 3

Stability of HKGs Expression in Mouse Liver under TCDD Exposure. A. The logarithmic values of ΔCt (log2^ΔCt), where ΔCt = Ct (TCDD) − Ct (control), are depicted for the 15 HKGs in the livers of mice exposed to TCDD compared to the control group. The horizontal line represents the median value. B. The stability ranking of HKGs determined by RefFinder across all study subjects. The stability of HKG expression was assessed using the BestKeeper computational tool. The most stable HKGs are indicated by an arrow pointing to the left, while the least stable HKGs are indicated by an arrow pointing to the right. Measurements were performed in triplicate for three cDNA preparations (n = 9).

Fig. 4

Stability of HKGs expression in mouse kidney as a function of TCDD Exposure A. Logarithmic values of 2^ΔCt, where ΔCt = Ct (TCDD) − Ct (control), are illustrated for the 15 HKGs in the kidneys of mice exposed to TCDD compared to the control group. The horizontal line denotes the median value. B. The stability ranking of HKGs determined by RefFinder across all study subjects. HKG expression stability was evaluated using the BestKeeper computational tool. The most stable HKGs are denoted by an arrow pointing to the left, while the least stable HKGs are indicated by an arrow pointing to the right. Measurements were conducted in triplicate for three cDNA preparations (n = 9).

Fig. 5

Stability of HKGs in mice lung as a function of TCDD exposure. A. The log2^ΔCt values, where ΔCt = Ct (TCDD) − Ct (control), for the 15 HKGs in the lungs of mice exposed to TCDD compared to the control group are presented. The horizontal line represents the median value. B. The stability ranking of HKGs determined by RefFinder across all study subjects. HKG expression stability was assessed using the BestKeeper computational tool. The most stable HKGs are indicated by an arrow pointing to the left, while the least stable HKGs are indicated by an arrow pointing to the right. Measurements were conducted in triplicate for three cDNA preparations (n = 9).

Fig. 6

Stability of Housekeeping Genes (HKGs) in Mouse Ovary under TCDD Exposure. A. Logarithmic values of 2^ΔCt, where ΔCt = Ct (TCDD) − Ct (control), for the 15 HKGs in the ovaries of mice exposed to TCDD compared to the control group are shown. The horizontal line indicates the median value. B. The stability ranking of HKGs determined by RefFinder across all study subjects. The stability of HKG expression was assessed using the BestKeeper computational tool. The most stable HKGs are denoted by an arrow pointing to the left, while the least stable HKGs are indicated by an arrow pointing to the right. Measurements were performed in triplicate for three cDNA preparations (n = 9).

Fig. 7

Stability of Housekeeping Genes (HKGs) in Mouse Testis under TCDD Exposure. A. The log2^ΔCt values for the 15 HKGs in the testes of mice exposed to TCDD compared to the control group are presented. The horizontal line represents the median value. B. The stability ranking of HKGs determined by RefFinder across all study subjects. The stability of HKG expression was evaluated using the BestKeeper computational tool. The most stable HKGs are depicted by an arrow pointing to the left, while the least stable HKGs are indicated by an arrow pointing to the right. Measurements were conducted in triplicate for three cDNA preparations (n = 9).