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. 2020 Jul 14:2020:8926120.
doi: 10.1155/2020/8926120. eCollection 2020.

Selection of Suitable Reference Genes for qPCR Gene Expression Analysis of HepG2 and L02 in Four Different Liver Cell Injured Models

Jiyu Chen  1   2 , Zhenzhen Bao  1 , Yanli Huang  2 , Zhenglong Wang  2 , Yucheng Zhao  3
Affiliations

Selection of Suitable Reference Genes for qPCR Gene Expression Analysis of HepG2 and L02 in Four Different Liver Cell Injured Models

Jiyu Chen et al. Biomed Res Int. .

Abstract

Quantitative real-time PCR (qPCR) has become a widely used approach to analyze the expression level of selected genes. However, owing to variations in cell types and drug treatments, a suitable reference gene should be selected according to special experimental design. In this study, we investigated the expression level of ten candidate reference genes in hepatoma carcinoma cell (HepG2) and human hepatocyte cell line (L02) treated with ethanol (EtOH), hydrogen peroxide (H2O2), acetaminophen (APAP), and carbon tetrachloride (CCl4), respectively. To analyze raw cycle threshold values (Cp values) from qPCR run, three reference gene validation programs, including Bestkeeper, geNorm, and NormFinder, were used to evaluate the stability of ten candidate reference genes. The results showed that TATA-box binding protein (TBP) and tubulin beta 2a (TUBB2a) presented the highest stability for normalization under different treatments and were regarded as the most suitable reference genes of HepG2 and L02. In addition, this study not only identified the most stable reference genes of each treatment, but also suggested that β-actin (ACTB), glyceraldehade-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), and beta-2 microglobulin (B2M) were the least stable reference genes in HepG2 and L02. This work was the first report to systematically explore the stability of reference genes in injured models of HepG2 and L02.

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Conflict of interest statement

The authors did not report any conflict of interest.

Figures

Figure 1
Figure 1
Expression levels of ten reference genes (ACTB, B2M, GAPDH, TUBB2a, HPRT1, SDHA, TBP, YWHAZ, CYC1, GUSB) in HepG2 (a) and L02 (b). Squares in the middle of the box represent the mean values, horizontal lines in the box represent the median, and whiskers represent the highest and lowest values.
Figure 2
Figure 2
Expression stability of the reference genes in HepG2 evaluated by geNorm M values represents the average expression stability. From left to right, the value of M decreased in turn, indicating the stability gradually increased. Smaller M value means higher stability. The control group, ethanol, hydrogen peroxide, acetaminophen, and carbon tetrachloride were abbreviated to WT, EtOH, H2O2, APAP, and CCl4, respectively.
Figure 3
Figure 3
Calculation of the optimal number of reference genes for quantitative analysis using geNorm. Pairwise variation (Vn/n + 1) of reference genes analyzed in different treatments was listed in (a, b) set the cut-off threshold value 0.15, calculating the optimal number of reference genes for precise quantitative in this work. WT, E100 (E200, E400), H100 (H200, H400, H800), A2.5 (A5, A10), and C0.05% (C0.1%, C0.2%, C0.4%), respectively, were the abbreviation for the control group, EtOH-100 mM (200 mM, 400 mM), H2O2-100 μM (200 μM, 400 μM, 800 μM), APAP-2.5 mM (5 mM, 10 mM, 20 mM), CCl4-0.05% (0.1%, 0.2%, 0.4%).
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