BACKGROUND: Fetal and placental sex influence a variety of developmental processes during prenatal life
including metabolism, growth, and the response to in utero insults. Additionally, the National Institute of Health's requirement that sex as a biological variable be included into proposal design necessitates the development of tools to investigate sex during embryonic and fetal life. Rodent models are insightful models in the study of sexual dimorphism due to large litter sizes, short gestation period, and frequency of use as an animal model. In this methods paper, we demonstrate a multiplex PCR method to determine sex in fetal rat tail snips and placentas. METHODS AND RESULTS: We designed primers for X-chromosome and Y-chromosome homologs, DDX3X and DDX3Y, and developed a single-step PCR protocol that can determine the presence of both genes in one reaction. We performed PCR on fetal tail snips and placentas to amplify DDX3X only in females or DDX3X and DDX3Y in males. The multiplex PCR and subsequent gel electrophoresis revealed that the presence of only DDX3X or both DDX3X and DDX3Y could be detected in fetal tissues. We used adult male rat testis as a positive control and confirmed that both DDX3X and DDX3Y could be detected in adult male tissues as well. CONCLUSION: This protocol provides an important method to determine genetic sex in tissues before the ability to visually determine sex, allowing for sex to be used as a biological variable in prenatal research using the rat model.