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Prostaglandin E2 Receptor Subtype EP2- and EP4-regulated Gene Expression Profiling in Human Ciliary Smooth Muscle Cells

ABSTRACT: Changes of genome-wide mRNA transcription levels of human ciliary smooth muscle (hCSM) cells were determined by treating hCSM cells in culture with 200 nM of either an prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. This was followed by competitive hybridization of fluorescent Cy3 or Cy5 labeled cRNA probes derived from the treated versus untreated control total RNA samples onto an Agilent Human Whole Genome Expression oligonucleotide microarray. Log 2 (LN) of the intra-slide ratios (RATIO, PRE_VALUE) of treated versus untreated samples was reported as VALUE in the sample files. Keywords: prostaglandin E2 receptor agonists, subtype EP2, subtype EP4, hCSM

ORGANISM(S): Homo sapiens

PROVIDER: GSE10621 | GEO | 2008/02/24

SECONDARY ACCESSION(S): PRJNA107681

REPOSITORIES: GEO

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Project description:Changes of genome-wide mRNA transcription levels of human ciliary smooth muscle (hCSM) cells were determined by treating hCSM cells in culture with 200 nM of either an prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. This was followed by competitive hybridization of fluorescent Cy3 or Cy5 labeled cRNA probes derived from the treated versus untreated control total RNA samples onto an Agilent Human Whole Genome Expression oligonucleotide microarray. Log 2 (LN) of the intra-slide ratios (RATIO, PRE_VALUE) of treated versus untreated samples was reported as VALUE in the sample files. Experiment Overall Design: Human Ciliary Smooth Muscle Cell Culture: Experiment Overall Design: Human ciliary smooth muscle (hCSM) cells were isolated from a female donor, received from the San Diego Eye BankÂ® (San Diego, CA) and cultured in DMEM with 10% fetal bovine serum and 0.5% penicillin/streptomycin according to the method reported previously by Woldemussie et al. Experiment Overall Design: For transcriptome analysis, cells were used for experiments starting at passage 4. Three independent experiments at consecutive cell passages were performed. For each experiment 0.6 x 106 cells were plated onto 15 cm dishes in MEM D-Valine medium (PromoCell, Germany) supplemented with 10% FCS, L-glutamine and antibiotics/antimycotics. Cells were grown to ~80% confluency for 6 days, followed by starvation in absence of FCS and presence of 100 nM of the cyclooxygenase inhibitor indomethacin, 20 Âµg/ml fatty acid-free BSA and 4 Âµg/ml transferrin for 17 hours. Cells were then exposed for 6 hours to 200 nM of either the EP2 agonist AGN210937 or the EP4 agonist AGN202280 (Allergan, Irvine, CA) or the corresponding amount of DMSO in the absence of FCS and presence of transferrin. Experiment Overall Design: Total RNA Extraction: Experiment Overall Design: Total RNA was isolated using a RNA isolation kit (RNAqueous; Ambion, USA) which was followed by DNase I treatment (Turbo DNA-free; Ambion) and purification, according to the manufacturerâ??s protocols. RNA was quantitated using a spectrophotometer ND-1000 (Nanodrop Technologies, USA). RNA quality was assessed regarding purity and stability using a Bioanalyser 2100 (Agilent Technologies, USA). Extracted total RNA aliquots were snap-frozen in liquid nitrogen and stored at -800C for single use. Experiment Overall Design: cRNA Labelling and Oligonucleotide Microarray Hybridization: Experiment Overall Design: Total RNA was linearly amplified and labelled with Cy3 or Cy5 using a low RNA input Fluor Linear Amp Kit (Agilent Technologies, USA). Internal RNA controls from Agilentâ??s RNA Spike-in Kit were included, comprised of mixtures of 11 in vitro synthesized transcripts derived from the Adenovirus E1A transcriptome at various ratios. The final cRNA concentration of 500 ng total RNA used was typically 590-650 ng/Î¼l and the Cy3- or Cy5-cytidine incorporation was 5.4 -6.0 pmol/Î¼g cRNA as determined using the Nanodrop spectrophotometer ND-1000. Experiment Overall Design: 3.5 Î¼g of Cy3 and Cy5 labeled cRNA (treated vs. untreated samples) were competitively hybridized to high-density DNA arrays from Agilent Technologies (Whole Human Genome Oligo Microarray 44K) for 17 hours at 650C according to the manufacturerâ??s protocol. Each set of three microarray experiments for triplicate analysis contained one dye swap experiment. All microarrays were scanned and the intensities normalized over background as well as to eliminate signal intensity-dependent bias from the ratio of the two channels (Lowess normalization) using a microarray scanner from Agilent Technologies including proprietary software. All microarray data sets were imported into the microarray data analysis software Genespring 7.3 (Agilent Technologies) followed by comparison of normalized intensities. Gene identities were updated according to Agilentâ??s latest list of gene annotations (G4112F, 07Feb07). Experiment Overall Design: Consistency of microarray data quality was assessed by monitoring performance of the spiked internal RNA controls (330 microarray spots/array) at various ratios (Supplementary Data, Fig. S1), by carrying out a global analysis of all absolute fluorescent intensity values, and by analysis of negative control spots (314 microarray spots/array) for determination of detection thresholds.

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Prostaglandin E2 Receptor Subtype EP2- and EP4-regulated Gene Expression Profiling in Human Ciliary Smooth Muscle Cells

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