The selection and identification of compound housekeeping genes for quantitative real‐time polymerase chain reaction analysis in rat fetal kidney

Abstract

During quantitative real‐time polymerase chain reaction (RT‐qPCR) data analysis, the selection of optimal housekeeping gene is necessary to ensure the accuracy of results. It is noteworthy that housekeeping genes commonly used in adult studies may not be applicable for fetus. However, the stability analysis of housekeeping gene in fetal kidney has not been reported. This study intends to screen the applicable compound housekeeping genes in rat fetal kidney. In this study, eight housekeeping genes used in kidney studies based on literature reports (GAPDH, ACTB, 18S, HPRT, YWHAZ, HMBS, PPIA, and TBP) were selected as the research object. Their expression levels in the rat fetal kidney in physiological condition and the intrauterine growth retardation (IUGR) model induced by prenatal dexamethasone exposure (PDE) (0.2 mg/kg·day from gestation Days 9 to 20) was measured. Furthermore, these eight housekeeping genes were used to conduct relative quantitative analysis of nephrin expression in the fetal kidney in PDE‐induced IUGR model, to compare the influence of choosing different housekeeping gene on data analysis of nephrin expression and to verify the reliability of selected compound housekeeping genes. In this study, stable housekeeping genes of fetal kidney tissues in PDE‐induced IUGR model were identified: ACTB, GAPDH, TBP, and HMBS for males; ACTB, YWHAZ, and GAPDH for females. Besides, our results suggest that ACTB + GAPDH were the best compound housekeeping genes for normalization analysis in male fetal kidney studies, and ACTB + YWHAZ in females. This study will provide an experimental evidence basis for the selection of housekeeping genes in the RT‐qPCR experiment in renal development toxicology‐related models.

Identification of biomarkers and sex differences in the placenta of fetal growth restriction

“…[29][30][31][32] We adopted the protocol that was used maturely in our cooperator by subcutaneously injected by 0.2 mg/kg/d from GD9 to GD20. 31,[33][34][35] Our in vitro experiment supported the vascular dysfunction by presenting the dilated vascular lumen and the reduced expression of vegfa, which was in accordance with the dataset analysis (Figure S3a-c). On the one hand, the dilated vascular lumen was proved to slow the blood flow, which brought less blood to the placenta, 1 as was shown in our images that blood cells in the dexamethasone-treated placenta were less than that of the control.…”

“…Dexamethasone, a synthetic glucocorticoid, has been extensively applied in clinical due to its anti‐inflammatory, anti‐shock, and anti‐allergy effects, which is widely adopted for FGR model building 29–32 . We adopted the protocol that was used maturely in our cooperator by subcutaneously injected by 0.2 mg/kg/d from GD9 to GD20 31,33–35 . Our in vitro experiment supported the vascular dysfunction by presenting the dilated vascular lumen and the reduced expression of vegfa , which was in accordance with the dataset analysis (Figure S3a–c).…”