Microarrays are a powerful method for the global analysis of gene or protein content and expression, opening up new horizons in molecular and physiological systems. This review focuses on the critical aspects of acquiring meaningful data for analysis following fluorescence-based target hybridisation to arrays. Although microarray technology is adaptable to the analysis of a range of biomolecules (DNA, RNA, protein, carbohydrates and lipids), the scheme presented here is applicable primarily to customised DNA arrays fabricated using long oligomer or cDNA probes. Rather than provide a comprehensive review of microarray technology and analysis techniques, both of which are large and complex areas, the aim of this paper is to provide a restricted overview, highlighting salient features to provide initial guidance in terms of pitfalls in planning and executing array projects. We outline standard operating procedures, which help streamline the analysis of microarray data resulting from a diversity of array formats and biological systems. We hope that this overview will provide practical initial guidance for those embarking on microarray studies.
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- Author: P Ghazal x
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T Forster, D Roy, and P Ghazal
M T Rae, D Niven, A Ross, T Forster, R Lathe, H O D Critchley, P Ghazal, and S G Hillier
The human ovarian surface epithelium (HOSE) is a common site of gynaecological disease including endometriosis and ovarian cancer, probably due to serial injury-repair events associated with successive ovulations. To comprehend the importance of steroid signalling in the regulation of the HOSE, we used a custom microarray to catalogue the expression of over 250 genes involved in the synthesis and reception of steroid hormones, sterols and retinoids. The array included a subset of non-steroidogenic genes commonly involved in pro-/anti-inflammatory signalling. HOSE cells donated by five patients undergoing surgery for non-malignant gynaecological conditions were cultured for 48 h in the presence and absence of 500 pg/ ml interleukin-1α (IL-1α). Total RNA was reverse-transcribed into biotin-labelled cDNA, which was hybridised to the array and visualised by gold-particle resonance light scattering and charge-coupled device (CCD) camera detection. Results for selected genes were verified by quantitative reverse-transcription PCR. In five out of five cases, untreated HOSE cells expressed genes encoding enzymes required for de novo biosynthesis of cholesterol from acetate and subsequent formation of C21-pregnane and C19-androstane steroids. Consistent with the inability of HOSE cells to synthesise glucocorticoids, oestrogens or 5α-reduced androgens de novo, CYP21, CYP19 and 5α-reductase were not detected. The only steroidogenic gene significantly up-regulated by IL-1α was 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). Other cytokine-induced genes were IL-6, IL-8, nuclear factor κB (NFκB) inhibitor α, metallothionein-IIA and lysyl oxidase: inflammation-associated genes that respond to glucocorticoids. The only steroidogenic gene significantly suppressed by IL-1α was 3βHSD1. Other genes suppressed by IL-1α were aldehyde dehydrogenase (ALDH) 1, ALDH 10, gonadotrophin hormone-releasing hormone receptor, peroxisome proliferation-activated receptor-binding protein (PPAR-bp) and nuclear receptor subfamily 2 group F member 2. These results define a steroidogenic phenotype of cultured HOSE cells and provide a limited expression profile for genes with associated signalling functions. IL-1α co-ordinately induces 11βHSD1 and a panel of glucocorticoid-regulated, inflammation-associated genes in HOSE cells, providing further evidence that cortisol generated by 11βHSD1 could participate in the local resolution of inflammation associated with ovulation.