GXB: Diferential gene expression in Trophoblast cell cultures

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Description

Diferential gene expression in Trophoblast cell cultures - GSE4100

Purpose

The syncytiotrophoblast develops as a result of the fusion of placental villous cytotrophoblast cells. These multinuclear cells are essential for fetal-maternal exchange and production of pregnancy-related hormones. Placental deficiency is considered a major risk factor associated with intrauterine growth restriction and preeclampsia. Better understanding of placental patho-physiology requires knowledge on gene expression regulation involved in trophoblast differentiation. Using the well-established in vitro trophoblast differentiation model, we have performed a microarray analysis on mRNA expression in trophoblast and syncytiotrophoblast cell cultures. Dramatic changes in gene expression patterns were detected during trophoblast differentiation. As many as 3524 novel and known genes are found to be up- or down regulated for more than 2-fold. Real-time PCR analysis of a group of genes confirmed the reliability of the microarray data. Overall, this study provided a global view on the molecular events underlying trophoblast differentiation. Subsequent characterization on regulation and function of the identified genes may lead to discovery of new pathways important for placental differentiation and function.Keywords: Trophoblast culture cells

Experimental Design

Affymetrix GeneChipTM Human Genome U133 Plus 2.0 microarrays were used for mRNA profiling. RNA samples were subject to Agilent analysis for quality controls. cDNA was prepared from 10 µg of RNA, quantified by spectrometry, and used as a template for the synthesis of biotinylated cRNA using RNA transcript labeling reagent. The quality of the cRNA probe was verified by gel electrophoresis as well as a pilot hybridization using the Test-3 arrays. Hybridization solution containing fragmented cRNA probes and control cRNA was supplemented with herring sperm DNA and bovine serum albumin. The probe solution was heated at 99° C for 5 min followed by incubation at 45° C for 5 min before use. Hybridization was carried out at 45° C for 16 hr with constant rotation at 60 rpm. The arrays were washed and stained with streptavidin-phycoerythrin. The GeneChip 5.0 (Affymetrix) program was used to scan and quantitatively document the hybridization signals. Compilation of candidate gene and calculation of changes were performed on SpotFire and Microsoft Excel programs. To minimize the false-positive conclusion, only genes satisfying the following two criteria were considered candidate regulation targets for further analysis: 1) The gene’s hybridization signal reached an absolute level that was significantly higher than that of the background (P<0.05); 2) The changes is more than 2-fold when compared to control group.

Platform

Affymetrix HG-U133_Plus_2

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Diferential gene expression in Trophoblast cell cultures - GSE4100

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