Panel of suitable reference genes and its gender differences of fetal rat liver under physiological conditions and exposure to dexamethasone during pregnancy

Highlights

• There are gender differences in the selection of reference genes.

• The panel of suitable reference genes are different under physiological and PDE models.

• The panel of suitable reference genes are vital for accurate experimental results.

Abstract

The panel of suitable reference genes in the fetal liver have not been reported. In this study, five commonly used reference genes (GAPDH, β-actin, Rn18 s, Rpl13a, and Rps29) were firstly selected as candidates. Bestkeeper, GeNorm, and NormFinder software were then used to screen out the panel of suitable reference genes of male and female fetal rat liver under physiological and prenatal dexamethasone exposure (PDE) conditions. Finally, we verified the reliability of the screened panel of reference genes by standardizing sterol regulatory element binding protein 1c (SREBP1c) expression with different reference genes. The results showed that GAPDH + Rn18 s and GAPDH + Rpl13a were respectively the panel of suitable reference genes in male and female rat fetal liver under the physiological model, while Rn18 s + Rps29 and GAPDH + Rn18 s were respectively under the PDE model. The results showed that different reference genes affected the statistical results of SREBP1c expression, and the screened panel of suitable reference genes under the PDE model had smaller intragroup differences, when compared with other reference genes under physiological and PDE models. In conclusion, we screened and determined that the panel of suitable reference genes were GAPDH + Rn18 s and Rn18 s + Rps29 in the male rat fetal liver under physiological and PDE models, while they were GAPDH + Rpl13a and GAPDH + Rn18 s in the females, and confirmed that the selection of the panel of suitable reference genes in the fetal liver had gender differences and pathological model specificity.

Introduction

Real-time fluorescence quantitative PCR (RT-qPCR) is widely used for relative quantitative gene expression detection due to its advantages of high sensitivity, strong specificity, and high throughput [1]. House-keeping genes (HKGs) refer to the genes that are essential for maintaining cell life and express relatively stable in tissue cells [2], and are often used as reference genes in RT-qPCR analysis to standardize target gene expression. Therefore, the stability of the reference gene for RT-qPCR determines the accuracy of the experimental results. However, the study found that the stability of some commonly used reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (β-actin) and 18S ribosomal RNA (Rn18 s) varied with tissue type, development stage, gender and research model [[3], [4], [5], [6], [7], [8]]. For this reason, researchers should screen appropriate reference genes according to the actual situation. In addition, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) suggests using multiple stable reference genes to make up panel of reference genes to improve the accuracy of experimental results [9]. Vandesompele et al. also have proved that a relatively large error occurred when using a single reference gene for standardization [10]. All the above findings suggest that screening panel of reference genes in various tissues and treatment conditions are essential to obtain accurate experimental results.

As the largest gland in the mammal, the liver is an essential organ for regulating many physiological functions such as protein synthesis and metabolism [11]. It is known that the liver undergoes a complex development process before it matures [12]. The composition, ratio, and gene expression of hepatocyte are constantly changing [13], which indicates that the expression stability of the reference genes may also be altered during liver development, resulting in some frequently-used reference genes that fail to provide an accurate reference. Accordingly, the screening and identification of panel of reference genes that are stably expressed in fetal liver tissues is the basis and prerequisite for studying fetal liver development. However, the selection of the panel of reference genes in the liver during the fetal period has never been reported.

Barker proposed the developmental origin of adult diseases and fetal programming as early as 1993 [14]. Subsequently, domestic and foreign scholars carried out a large number of studies on the correlation of the adverse environment during pregnancy, fetal birth weight and various diseases, and proposed a new concept “developmental origin of health and disease” (DOHaD) in 2003 [15]. Numerous studies have shown that a variety of adverse environments during pregnancy (such as xenobiotic exposure, malnutrition, infection, oxidative stress) can increase intrauterine maternal glucocorticoid levels [[16], [17], [18], [19], [20], [21]]. Our previous studies also confirmed that the exposure of caffeine, nicotine, and ethanol during pregnancy could cause fetal-maternal glucocorticoid overexposure, accompanied by intrauterine growth retardation (IUGR) and multiple organ toxicities (such as liver dysplasia), which was related to the adaptive developmental programming of fetuses [[22], [23], [24], [25]]. Therefore, overexposure to fetal-maternal glucocorticoids in adverse environments during pregnancy may be an universal phenomenon. Dexamethasone, as a synthetic glucocorticoid, is widely used to prevent premature delivery and promote fetal lung maturation [26]. Increased studies have shown that IUGR induced by prenatal dexamethasone exposure (PDE) may increase offspring’s susceptibility to many diseases (such as cholestatic liver injury, and fatty liver). So, PDE has become a commonly used model for the study of liver development-related diseases [27,28]. The model is not only used to study the fetal developmental toxicity of dexamethasone, but also well simulates the process of low-exposure to endogenous glucocorticoids in the offspring during adverse pregnant environment. Therefore, screening the panel of suitable reference genes of fetal liver tissues under the PDE model can represent the change rules of the panel of reference genes’ stability to some extent in pathological liver development, and further improve the accuracy of research on liver development-related diseases.

In this study, we first selected five reference genes (GAPDH, β-actin, Rn18 s, Rpl13a, and Rps29) as candidates from the literature [29], and detected their expression by RT-qPCR in fetal rat liver tissues under physiological and PDE model conditions. Next, the stability of the candidate reference genes expression was analyzed by BestKeeper, GeNorm, and NormFinder software [30], and the panel of suitable reference genes were selected. Finally, we standardized the expression of sterol regulatory element binding protein 1c (SREBP1c) in the fetal liver under the PDE model to verify the screened panel of reference genes’ reliability. This study provides an experimental and theoretical basis for studies related to liver development.