Identification of appropriate reference genes for qPCR analyses of porcine placentae and endometria, supplying foetuses of different size and sex, at multiple gestational days

Abstract

Recent studies suggest associations exist between foetal size and sex, and gene expression at the porcine feto-maternal interface. It is essential to identify reference genes which have stable expression throughout gestation in feto-placental units associated with foetuses of different size and sex. qPCR was performed for 11 genes within porcine placentae and endometria at gestational days (GD) 30, 60 and 90. Several reference genes were found to have stable expression in these samples. The combination of B2m1 and Tbp1, and Hprt1 and Tbp1 had the most stable expression in endometria and placentae, respectively. Reference genes identified as having stable expression were utilized in a larger experiment with placentae and endometria associated with foetuses of different size and sex at four GD. The average expression of B2m1 and Tbp1 mRNAs was suitable for the normalization of temporal changes in endometria, and comparison between endometria supplying foetuses of different size throughout gestation. The average expression of Hprt1 and Tbp1 mRNAs was suitable for the normalization of placental mRNA expression for comparison of temporal changes and sex differences between placentae supplying foetuses of different sex throughout gestation. This combination was suitable for the normalization of mRNA expression in placentas supplying GD30, GD60 and GD90 foetuses of different size. This study has identified reference genes with stable expression in placentae and endometria across multiple gestational days, in tissues associated with foetuses of different size and sex. The results of these experiments highlight the importance of selecting appropriate reference genes for the biological comparison under investigation.