Housekeeping gene expression stability in reproductive tissues after mitogen stimulation

BACKGROUND: Intrauterine infection during pregnancy can trigger a local inflammatory response leading to several complications, such as preterm labor. Many studies have used in vitro and in vivo models employing mitogens to induce the expression of the characteristic proinflammatory mediators triggered by infection. However, relative expression assays depend on the stability of housekeeping gene expression, which can vary depending on certain stimuli. In this study, we analyzed the stability and pairwise variation in the expression of GAPDH, ACTB and RNA18S1 in cultured reproductive tissues under mitogen stimulation. We used fetal membranes, placental villous and umbilical cord explants from patients with normal term pregnancies (>37 weeks of gestation), as well as myometrium and cervix explants from patients undergoing hysterectomies. Tissues were stimulated with lipopolysaccharide or phytohemagglutinin for 24 hours. We then analyzed the expression stability and the pairwise variation of GAPDH, ACTB and RNA18S1 from real time quantitative RT-PCR absolute threshold cycles (Cp) using geNorm software. RESULTS: In all of the tissues, the three housekeeping genes showed great stability under our experimental conditions. Pairwise variation analyses showed that only two reference genes are required for adequate normalization, GAPDH and ACTB being optimal in the cervix, fetal membranes and umbilical cord, while GAPDH and RNA18S1 are best for normalization in the placenta and myometrium. CONCLUSION: Our results show that GAPDH, ACTB and RNA18S1 are adequate references for gene expression normalization in reproductive tissues stimulated with mitogens in culture.