Investigation Title Transcription profiling of human intestinal (Caco-2) and placental (JAR) cell-lines, after 12h or 24h exposure to different zinc concentrations to examine Zinc-regulated gene expression Comment[Submitted Name] Ford A107custom array_Caco-2 and JAR_Zinc treatment Experimental Design compound_treatment_design co-expression_design in_vitro_design dye_swap_design transcription profiling by array Experimental Design Term Source REF mo mo mo EFO Comment[ArrayExpressReleaseDate] 2008-03-20 Comment[AEMIAMESCORE] 5 Comment[ArrayExpressAccession] E-MEXP-1064 Comment[MAGETAB TimeStamp_Version] 2010-08-09 19:41:56 Last Changed Rev: 13058 Experimental Factor Name dose cell line compound time Experimental Factor Type dose cell_line compound_treatment_design time Experimental Factor Term Source REF Person Last Name Jackson Person First Name Kelly Person Mid Initials A Person Email k.a.jackson@ncl.ac.uk Person Phone 0191 2226000 (x8355) Person Fax Person Address Medical School, Framlington Place, Newcastle-upon-Tyne, Tyne and Wear, NE2 4HH, UK Person Affiliation Institute for Cell and Molecular Biosciences Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type technical_replicate spike_quality_control dye_swap_quality_control Quality Control Term Source REF mo The MGED Ontology Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-03-20 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description Zinc-regulated gene expression in intestinal (Caco-2) and placental (JAR) cell-lines, after 12h or 24h exposure to different zinc concentrations Protocol Name P-MEXP-50534 P-MEXP-49854 P-MEXP-50535 P-MEXP-49853 P-MEXP-49867 P-MEXP-49866 P-MEXP-49868 P-MEXP-49855 P-MEXP-49856 P-MEXP-49857 P-MEXP-49865 P-MEXP-49858 P-MEXP-49859 P-MEXP-50568 Protocol Type specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action grow grow grow grow pool nucleic_acid_extraction labeling labeling hybridization feature_extraction bioassay_data_transformation Protocol Description Remove media from cells, replace with basal medium (containing ~3 microM zinc). Remove media from cells, replace with basal medium supplemented to 12 microM zinc with ZnCl2 (purchased from Sigma). Remove media from cells, replace with basal medium supplemented to 100 microM zinc with ZnCl2 (purchased from Sigma). JAR monolayers (passage 10) grown to confluency (5-7 d post-seeding) in six replicate 75cm2 flasks. Media replaced every 2-3 d. Caco-2 cells (passage ??) were grown for a total of 14 d post-seeding (7 d post-confluency) to form differentiated monolayers. Media replaced every 2-3 d. JAR monolayers (passage 10) grown to confluency (5-7 d post-seeding) in six replicate 75cm2 flasks. Media replaced every 2-3 d. Caco-2 cells (passage ??) were grown for a total of 14 d post-seeding (7 d post-confluency) to form differentiated monolayers. Media replaced every 2-3 d. Extracted total RNA from six replicate flasks then pooled Total RNA extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. 100 micrograms RNA was DNase-treated using 20 U DNase enzyme (Roche), then purified by phenol:chloroform extraction. CyScribe post-labelling kit (Amersham Biosciences) was used to sythesize cDNA from 20 micrograms DNase-treated RNA. Two-step process using CyScribe post-labelling kit (Amersham Biosciences): 1) Incorporation of amino allyl-dUTP (aa-dUTP) during reverse transcription of the RNA. Reverse transcription reactions contained 20 micrograms total RNA. Amino allyl-labelled cDNAs purified using GFX columns (Amersham Biosciences) according to the manufacturer's instructions. 2) Chemical labelling of amino allyl modified cDNA using Cy3 NHS-esters (Amersham Biosciences). Cy3-labelled cDNAs purified using GFX columns. Two-step process using CyScribe post-labelling kit (Amersham Biosciences): 1) Incorporation of amino allyl-dUTP (aa-dUTP) during reverse transcription of the RNA. Reverse transcription reactions contained 20 micrograms total RNA. Amino allyl-labelled cDNAs purified using GFX columns (Amersham Biosciences) according to the manufacturer's instructions. 2) Chemical labelling of amino allyl modified cDNA using Cy5 NHS-esters (Amersham Biosciences). Cy5-labelled cDNAs purified using GFX columns. Pre-hybridised with hybridisation buffer (3x SSC, 0.15% SDS) for a total of 90 s at 42oC. Probes were injected when prompted by the hyb chamber. Hybridisation at 42oC for 16 h with low agitation. Washes: 1) 30oC for 2 x 30 s (2x SSC, 0.1% SDS) 2) 30oC for 2 x 30 s (1x SSC) 3) 30oC for 2 x 30 s (0.5x SSC) Slides were dried using compressed N2 gas. Slides were scanned with a GenePix 4000B confocal laser scanner (Axon Instruments), using laser excitation at 635nm for Cy3 and 532nm for Cy5. Photo-multiplier tube (PMT) gain was optimised for each slide so that detection of low and high intensity signals was balanced and saturation tolerance was set at 0.005% to avoid pixel saturation. GenePix Pro 6.0 (Molecular Devices, USA) was used to extract raw data from the images. Local background subtraction was used, whereby a background value is calculated for each feature individually, based on the intensity of pixels immediately surrounding the feature. In addition, individual features on each array slide were inspected visually and poor quality features were flagged as bad. Two hybridisations (dye swaps) were performed for each test treatment vs. control treatment comparison, each with genes spotted in duplicate. Therefore for each gene data is calculated based on 4 replicate spots. To account for dye swap the signal and control channel measurements were reversed for these hybridisations. The raw data were processed in the following way to yield transformed data: 1) Filter low quality spots 2) Generate normalised expression ratios 3) Calculate average expression ratios 1) Filter low quality spots: For each hybridisation a cut-off was applied to discard data from weak signals. The background cut-off was equal to 2 SD above the mean intensity value of the negative control spots on the array (different species – mwgaracontrol#002). Spots were classified as ‘not present’ if they: a) had intensity values in both channels below the cut-off, or; b) were flagged as bad in the image analysis software GenePix Pro (flag in GenePix raw data files -100). 2) Generate normalised expression ratios: Expression ratios were generated for each spot by dividing intensity in the signal channel by intensity in the control channel (i.e. Cy5/Cy3 or Cy3/Cy5 for dye swaps). Expression ratios were corrected using factors calculated from the median intensity ratios of: a) internal standards in the control and test samples (spike control – mwgaracontrol#001); b) eight housekeeping genes on the array (mwghuman10K#1000-1008). Only those spots marked ‘present’ (i.e. passed the filter low quality spots criteria) were used to calculate correction factors. 3) Calculate average expression ratios and p-values: For each test treatment vs. control treatment comparison an average signal/control ratio was calculated for each gene using the GeneSpring GX 7.3 package (www.agilent.com). Ratio values greater than 1 represent higher expression in test treatment compared to control treatment. Values less than 1 represent lower expression in test treatment. Where ‘no data’ is displayed spots were not present in any of the replicates (i.e. did not pass the filter low quality spots criteria). Student’s t-tests were performed by GeneSpring for each gene and p-values are displayed. Where ‘no replicates’ is displayed p-values could not be calculated due to spots being not present in two or more replicates. Protocol Parameters min temperature;media;start time; min temperature;start time;media; min temperature;media;start time; start time;min temperature;media; Amplification;Extracted product; Amplification;Amount of nucleic acid labeled;Label used; Amount of nucleic acid labeled;Label used;Amplification; time;Quantity of label target used;temperature;Volume;Chamber type; Protocol Hardware GenePix 4000B [Axon Instruments] Protocol Software GenePix Pro [Axon Instruments] Protocol Contact Protocol Term Source REF mo mo mo The MGED Ontology mo mo The MGED Ontology The MGED Ontology SDRF File E-MEXP-1064.sdrf.txt Term Source Name The MGED Ontology ArrayExpress mo The MGED Ontology EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version