Selection and validation of reference genes for gene expression studies of decidualization based on RNA sequencing

Decidualization is a multistep and complex physiological process used to aid the development of an implanting embryo. To date, the potential genes regulating decidualization have not been elucidated. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in gene expression studies, with relative quantification being the predominant method due to its simplicity, cost-effectiveness, and lower sample requirements. This method determines gene expression levels by normalizing to reference genes. However, the selection of stable reference genes for studies on decidualization remains a challenge. Based on the RNA-seq dataset from human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs), ten new candidate reference genes were identified. The expression of these ten candidates, along with the commonly used reference gene beta-actin, was measured in ESCs, DESCs, and decidual stromal cells (DSCs) through RT-qPCR. Five algorithms were used to systematically identify suitable reference genes. The results indicated that Staufen doublestranded RNA binding protein 1 (STAU1) was most stable for induced decidualization in vitro, showing consistent expression in ESCs and DSCs. Using STAU1 as the reference gene, the expression levels of insulin like growth factor binding protein 1 and prolactin in DESCs were significantly higher than those in ESCs. Stau1 was further validated with both natural pregnancy and artificially induced decidualization mouse models. Based on our bioinformatics analysis, we also propose that kelch like family member 9 and TSC complex subunit 1 may serve as additional reference genes. Our findings offer valuable insights for gene expression studies of endometrial decidualization.