ABSTRACT: Comparison of gene expression in labouring and non-labouring myometrium. In this work, gene expression in 48 (22 labouring, 26 non-labouring) myometrial samples was measured using Illumina HT-12 v4.0 BeadChips. Biopsy samples of full thickness lower segment specimens of human myometrium were selected from the Edinburgh Reproductive Tissue Biobank (ERTBB, http://www.crh.ed.ac.uk/biobank/). All tissue samples were collected during Caesarean section from participant tissue donors with informed and written consent, according to the ethical approval and governance granted to the Edinburgh Reproductive Tissues BioBank by the West of Scotland Research Ethics Committee 4 (09/S0704/3). In order to maximise biological replicates on the array, all 22 labouring samples that fulfilled our inclusion criteria and that were stored in the ERTBB as of August 2012 were selected. In order to minimise inter-group variation in baseline characteristics, a second group of non-labouring samples (n=26) were selected by matching to labouring samples on maternal BMI, gestational age, parity and maternal age. We did not exclude smokers or women who had received medication to induce labour. We restricted the number of non-labouring samples to 26 so that all samples (n=48) could fit on three array chips. Forty-five of the samples had been transferred into RNAlater® (Life Technologies, Invitrogen, Carlsbad, CA, USA) immediately after collection and then stored at -80°C. Three labouring samples had not been transferred to RNAlater® before freezing but were also included in the array analysis in order to maximise the number of labouring samples. Total RNA was isolated using TRI Reagent® and the Qiagen RNeasy Lipid Tissue kit protocol (Qiagen, Valencia, CA, USA). The quantity and quality of RNA was assessed using a NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). RNA quality was further assessed in the biotin-labelled samples by the Wellcome Trust Clinical Research Facility, Edinburgh using a Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE, USA). To prepare samples for the microarray experiment, 450ng total RNA was amplified and biotin-labelled using the Illumina® TotalPrepTM RNA Amplification Kit (Ambion, Austin, TX, USA). Samples were split randomly over four Illumina HT-12 v4.0 BeadChips to minimise any effect of inter-chip variability. One sample was used per well. The chips were imaged using a BeadArray Reader and raw data was obtained with Illumina BeadStudio software. Raw data were preprocessed using the Lumi package in R version 2.14.1. The data was subjected to Robust Multichip Average (RMA) background correction before quantile normalisation to remove non-biological systematic variation.
