Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR.

A growing need exists to consider fetal sex as a biological variable and accurately assess sex-specific effects. Among the multiple methods used to determine fetal sex, quantitative real-time polymerase chain reaction (qRT-PCR) of (sex-determining region Y) with genomic DNA (gDNA) is commonly used in addition to use of methodologies such as transcriptomics and detection of Barr body. However, messenger RNA (mRNA), a product of gDNA, has not been previously assessed for sex determination. Using placental samples from timed-pregnant Wistar rats at gestational day (GD) 16, this study assessed the compatibility of detection using gDNA versus mRNA to determine fetal sex. Samples used in this current study come from a larger study that investigated trichloroethylene (TCE) reproductive toxicity and potential modulation by N-acetyl-L-cysteine (NAC) and aminooxyacetic acid (AOAA). In 90 out of 91 samples, the sex classification determined by gDNA matched the sex classification determined by mRNA analyzing (/) values. For both gDNA and mRNA, statistically significant differences in / values between males and females were observed with samples considered in totality and when samples were separated by treatment groups (all comparisons were <0.01 or below, and all but two comparisons were <0.001 or below). Finally, the validity of using Cq values to determine fetal sex and the reference gene were also discussed. Together, this study suggests that determination of fetal sex in Wistar rats can be accomplished using measurements in gDNA or mRNA with highly compatible results.