Gene expression profile in labouring and non-labouring human placenta near term

Sample and RNA preparation: The series is composed of seventeen hybridizations for analysis of differentially expressed genes in placenta tissues from women after vaginal delivery (labouring) and after elective caesarean section (non-labouring). Total RNA extraction was performed by using the MagNa Pure Compact RNA Isolation Kit (Roche Applied Science). Labeling and hybridization: Total RNA was reverse transcribed and labeled with Cy3-attached dendrimer using the Genisphere 3DNA HS kit (Genisphere) as described in the manufacturer’s protocol. Hybridizations were carried out with a TECAN HS4800 instrument using the foramamide-based hybridization buffer from Genisphere containing 5% dextran sulphate and 0.5 microgram COT1 DNA/microgram RNA at 37°C for 23 hours. 3DNA dendrimer hybridizations were carried out in formamide-based hybridization buffer alone. Post-hybridization washes were carried out at room temperature with 2xSSC for 1 min, 0.2% SDS/2xSSC for 1 min and finally with0.2xSSC for 30 sec. Scanning, image analysis and data processing: The microarrays were scanned on an AXON 4000B scanner and images were quantified using GenePix pro 6.0 software. The background estimates were calculated using the morphological opening method. Spots that displayed a signal-to-noise ratio of less than 3 or that were significantly saturated (more than 20 % saturation among foreground pixels) were filtered out. The median was used as the averaging measure of the foreground pixels. After quality control, genes that were present in less than 50 % of the arrays were filtered out. Normalization was carried out using lowess normalization Sample labelling was balanced across the groups, presumably attenuating any dye-effect. For the purpose of finding differentially expressed genes empirical Bayes analysis, using the LIMMA package was applied. The data were analysed using a two-component linear model. The prior guess of the number of D.E genes was set to 0.01. Multiple testing was accounted for by estimating the false discovery rate. We applied the Benjamini-Hochberg procedure, as well as Storeys less conservative Q-value procedure was applied. In none of the cases were any genes significant at a reasonable (15-20% FDR) level. In summary, a 2.5-fold change in expression of any gene that was present on ≥ 8 arrays was considered significant, if the difference in fluorescence intensity between two groups reached a p-value of < 0.01. Keywords: Tissue samples of placenta from women after vaginal delivery (labouring) and after elective caesarean section (non-labouring) Samples from the placenta of 17 women who had elective sectio(non-labouring) were hybridised together on the same array with placenta samples from women who had vaginal delivery (labouring). This resulted in 17 direct comparisons on 17 arrays. To reduce the dye-bias, every second array had a reversed labeling orientation.