ONTOLOGY SOURCE REFERENCE Term Source Name "CHEBI" "OBI" "BTO" "NCBITAXON" "XCO" Term Source File "http://data.bioontology.org/ontologies/CHEBI" "http://data.bioontology.org/ontologies/OBI" "http://data.bioontology.org/ontologies/BTO" "http://data.bioontology.org/ontologies/NCBITAXON" "http://data.bioontology.org/ontologies/XCO" Term Source Version "78" "21" "20" "2" "33" Term Source Description "Chemical Entities of Biological Interest Ontology" "Ontology for Biomedical Investigations" "BRENDA Tissue and Enzyme Source Ontology" "National Center for Biotechnology Information (NCBI) Organismal Classification" "Experimental Conditions Ontology" INVESTIGATION Investigation Identifier "Inv_001" Investigation Title "Investigation" Investigation Description "" Investigation Submission Date "" Investigation Public Release Date "" Comment [Created with configuration] "" Comment [Last Opened With Configuration] "" Comment[Created With Configuration] "" Comment[Last Opened With Configuration] "" INVESTIGATION PUBLICATIONS Investigation PubMed ID "" Investigation Publication DOI "" Investigation Publication Author List "" Investigation Publication Title "" Investigation Publication Status "" Investigation Publication Status Term Accession Number "" Investigation Publication Status Term Source REF "" INVESTIGATION CONTACTS Investigation Person Last Name "" Investigation Person First Name "" Investigation Person Mid Initials "" Investigation Person Email "" Investigation Person Phone "" Investigation Person Fax "" Investigation Person Address "" Investigation Person Affiliation "" Investigation Person Roles "" Investigation Person Roles Term Accession Number "" Investigation Person Roles Term Source REF "" STUDY Study Identifier "GSE21274" Study Title "Expression profiling of genes responding to flurbiprofen enantiomers in HaCaT cells" Study Description "Previous work from our laboratory has identified R-flurbiprofen as a candidate chemopreventive agent for skin cancer. Despite our findings, and the investigation of R-flurbiprofen as a chemopreventive for prostate and colon cancer by other groups, the mechanism of action remains unclear. To gain mechanistic insight into these anti-proliferative effects, we carried out microarray analysis of HaCaT cells in response to both R-flurbiprofen and S-flurbiprofen treatment. This analysis shows that both R-flurbiprofen and S-flurbiprofen produced significant changes in gene expression compared to untreated control, however, no significant differences were identified between effects induced by the individual enantiomers. Many of the genes found to be altered compared to control are implicated in cellular growth, apoptosis and metastasis, a selection of which are discussed. The altered expression of these genes may contribute to the anti-proliferative effects of R-flurbiprofen and S-flurbiprofen. Keywords: oligonucleotide array, HaCaT, flurbiprofen, NSAID The study was designed as a triangular comparison, where RNA samples from both R-flurbiprofen and S-flurbiprofen treated cells would be compared to samples from untreated control cells to identify genes which were altered by the drugs. Additionally, direct comparison between samples from R-flurbiprofen treated cells and S-flurbiprofen treated cells would be carried out to facilitate identification of genes with different regulation between the two enantiomers. The Compugen 19k Human Oligo array was used to carry out this expression profiling. Array printing, sample labeling, hybridisation and data analysis were carried out by Adelaide Microarray Centre, where the Spot software package was used for data extraction and analysis. 3 arrays were used to compare R-flurbiprofen extracts to untreated controls, while 2 arrays each were used for comparison of S-flurbiprofen to control, and R-flurbiprofen to S-flurbiprofen." Comment[Study Grant Number] "" Comment[Study Funding Agency] "" Study Submission Date "2010-04-08" Study Public Release Date "2010-05-09" Study File Name "s_GSE21274.txt" STUDY DESIGN DESCRIPTORS Study Design Type "" Study Design Type Term Accession Number "" Study Design Type Term Source REF "" STUDY PUBLICATIONS Study PubMed ID "" Study Publication DOI "" Study Publication Author List "" Study Publication Title "" Study Publication Status "" Study Publication Status Term Accession Number "" Study Publication Status Term Source REF "" STUDY FACTORS Study Factor Name "compound" Study Factor Type "compound" Study Factor Type Term Accession Number "" Study Factor Type Term Source REF "" STUDY ASSAYS Study Assay File Name "a_GSE21274_transcription_profiling_DNA_microarray.txt" Study Assay Measurement Type "transcription profiling" Study Assay Measurement Type Term Accession Number "http://purl.obolibrary.org/obo/OBI_0000424" Study Assay Measurement Type Term Source REF "OBI" Study Assay Technology Type "DNA microarray" Study Assay Technology Type Term Accession Number "http://purl.obolibrary.org/obo/OBI_0400148" Study Assay Technology Type Term Source REF "OBI" Study Assay Technology Platform "GPL10289" STUDY PROTOCOLS Study Protocol Name "RNA extraction" "labeling" "nucleic acid hybridization" "data collection" "normalization data transformation" "data transformation" "sample collection" Study Protocol Type "RNA extraction" "labeling" "nucleic acid hybridization" "data collection" "normalization data transformation" "data transformation" "sample collection" Study Protocol Type Term Accession Number "" "" "" "" "" "" "" Study Protocol Type Term Source REF "" "" "" "" "" "" "" Study Protocol Description "Pelletted cells are dissolved in 500無 Trizol then 100無 of chloroform is added and the mixture is vortexed, left on ice for 15min, then centrifuged (6500 x g ) for 30min. at 4C. The upper aqueous phase is retained, mixed with an equal volume of 70% ethanol (in DEPC H2O) then the RNA is further purified using a Qiagen RNeasy kit. Briefly, the RNA/ethanol mixture is applied to a RNeasy column then centrifuged at 6500 x g for 1min. The eluent is re-passed through the column which is then washed with 700無 of buffer RW1. The column is washed twice with 500無 of buffer RPE then residual buffer is removed by spinning the column at 6500 x g for 1min. The purified RNA is eluted into a clean tube with 30無 of DEPC treated water. The RNA was then concentrated by precipitation. Add 1/10 volume of 3M sodium acetate (pH=5.2) and 2 volumes of ethanol. The mixture is cooled to -80C for 30min then RNA is pelletted by spinning (6500 x g ) for 20min. at 4C. The pellet is washed by adding 500無 of ethanol and spinning at (6500 x g ) for 10min. The supernatant is decanted and the pellet is left to dry for 60min. at room temperature. The RNA is dissolved in DEPC treated water and the concentration is determined. Aliquots of 30痢 in 20無 of DEPC H2O were used for labeling and hybridisation." "2無 of anchored polyT(V)N (2痢/無) is added and the RNA aliquot is incubated at 70C for 10min. The sample is then placed on ice. The sample is mixed with 6無 of 5X Superscript II buffer, 2無 of 0.1M DTT, 2無 of Superscript II (200U/無) and 0.6無 of aminoallyl (aa) dNTP mix (25mM dATP, 25mM dGTP, 25mM dCTP, 10mM dTTP and 15mM aa dUTP) then incubated at 42C for 2.5 hours. The RNA is hydrolysed by adding 10無 of 0.25M NaOH, 10無 of 0.5M EDTA (pH=8) and incubating the mix at 65C for 15min. The reaction is neutralised by adding 15無 of 0.2M acetic acid. The cDNA is purified using a QIAquick PCR purification kit. Briefly, the cDNA is mixed with 300無 of buffer PB then applied to the Qiagen column and centrifuged at 6500 x g for 1min. The eluent is re-passed through the column. The column is washed twice with 600無 of buffer PE then residual buffer is removed by spinning the column at 6500 x g for 1min. The sample is eluted into a clean tube with 90無 of Milli Q water. The purified cDNA is dried under reduced pressure then dissolved in 9無 of 0.1M NaHCO3 (pH 9.0). The mixture is added to a Cy dye aliquot*, mixed then left to incubate at room temperature for 60min. in the dark. The labeled cDNA is mixed with 41無 of Milli Q water then purified using a QIAquick PCR purification kit. Briefly, the cDNA is mixed with 250無 of buffer PB then applied to the Qiagen column and centrifuged at 6500 x g for 1min. The eluent is re-passed through the column. The column is then washed twice with 600無 of buffer PE then residual buffer is removed by spinning the column at 6500 x g for 1min. The sample is eluted into a clean tube with 90無 of Milli Q water. The purified labeled cDNA samples appear purple after being dried under reduced pressure. *One Cy dye sachet (Amersham cat#PA23001 and PA25001) is dissolved in 73無 of anhydrous DMSO then 4.5無 aliquots are distributed into sealable tubes. Each aliquot is dried under reduced pressure, sealed then stored in a desiccator at -20C." "Carried out by the Adelaide Microarray Centre Prior to hybridization each microarray is immersed in 50 ml of hot Milli-Q water (80-95C) and agitated gently for 5 min. The slide is then dried by centrifugation at 750rpm for 5min. The labeled cDNA is mixed with 0.64?L of 25mg/mL yeast tRNA, 4?L of 2mg/mL poly A and 20?L of 1mg/mL Cot-1 DNA. The mix is dried under reduced pressure then dissolved in the appropriate volume of formamide and 6.25 X SSC The mixture is heated to 100C for 3min., transferred directly to ice then 0.5?L of 10% SDS is added before the solution is applied to the centre of the cover slip. The array is lowered onto the cover slip then incubated at 42C overnight in a humidified chamber. Following incubation, the array is immersed in solution A Solution A (0.5 X SSC, 0.01% SDS) until the coverslip disengages the surface. After the coverslip is discarded the array is washed in Solution A for 5 min., Solution B (0.5 X SSC) for 5 min then Solution C (0.2 X SSC) for 3 min. The slide is dried in a centrifuge at 750 rpm for 5 min and stored in the dark prior to scanning." "Carried out by the Adelaide Microarray Centre. " "Carried out by the Adelaide Microarray Centre. Images analysed and data extracted using the Spot Software package (CSIRO Australia). Background correction was applied to mean spot intensities. Ratios were determined based on the spot intensities, with the ratio then normalised. Linear regression was used to identify statistical significance." "" "Confluent HaCaT cells were treated with 1mM R-flurbiprofen, 1mM S-flurbiprofen, or no drug dissolved in RPMI-1640 media, 1x GlutaMax-1, 10% FBS for 4 hours." Study Protocol URI "" "" "" "" "" "" "" Study Protocol Version "" "" "" "" "" "" "" Study Protocol Parameters Name "" "" "" "" "" "" "" Study Protocol Parameters Name Term Accession Number "" "" "" "" "" "" "" Study Protocol Parameters Name Term Source REF "" "" "" "" "" "" "" Study Protocol Components Name "" "" "" "" "" "" "" Study Protocol Components Type "" "" "" "" "" "" "" Study Protocol Components Type Term Accession Number "" "" "" "" "" "" "" Study Protocol Components Type Term Source REF "" "" "" "" "" "" "" STUDY CONTACTS Study Person Last Name "" Study Person First Name "" Study Person Mid Initials "" Study Person Email "" Study Person Phone "" Study Person Fax "" Study Person Address "" Study Person Affiliation "" Study Person Roles "" Study Person Roles Term Accession Number "" Study Person Roles Term Source REF ""