Evaluation of reference genes for expression studies in leukocytes from term human pregnancy

Abstract

IntroductionHuman labor is considered an inflammatory process modulated by systemic and local leukocytes that infiltrate into the maternal–fetal interface. The putative roles of these leukocytes are currently being studied with gene expression assays. Such assays are normalized by the expression of housekeeping genes. However, expression of housekeeping genes may vary depending on the cell type and/or the experimental conditions. The aim of this study was to analyze the expression stability of several housekeeping genes in leukocytes from term human pregnancies, considering both anatomical origin and presence of labor. MethodsWe analyzed the gene expression of ACTB, B2M, GAPDH, GUSB, PGK1, RN18S1, TBP and UBC in leukocytes from maternal peripheral blood, placental blood and choriodecidua in women delivering at term with or without the presence of labor through real-time qPCR. Then we used geNorm to evaluate expression stability and pairwise variation. ResultsThe expression of all tested genes showed to be stable independent of the anatomical compartment and the absence or presence of labor. However, PGK1, GUSB and TBP showed to be the most stable and RN18S1 the least stable. Pairwise variation analyses showed that only two genes are needed for normalization yet the inclusion of a third improves its accuracy. DiscussionPGK1, GUSB and TBP are the most adequate reference genes for gene expression normalization in leukocytes from term pregnancies, regardless of their anatomical origin (maternal peripheral blood, placental blood or choriodecidua) or the presence or absence of labor. Our study is the first report on housekeeping gene stability in leukocytes from healthy pregnant women.