ONTOLOGY SOURCE REFERENCE
Term Source Name	"CHEBI"	"BTO"	"NCBITAXON"	"XCO"	"UO"	
Term Source File	"http://data.bioontology.org/ontologies/CHEBI"	"http://data.bioontology.org/ontologies/BTO"	"http://data.bioontology.org/ontologies/NCBITAXON"	"http://data.bioontology.org/ontologies/XCO"	"http://data.bioontology.org/ontologies/UO"	
Term Source Version	"78"	"20"	"2"	"33"	"42"	
Term Source Description	"Chemical Entities of Biological Interest Ontology"	"BRENDA Tissue and Enzyme Source Ontology"	"National Center for Biotechnology Information (NCBI) Organismal Classification"	"Experimental Conditions Ontology"	"Units of Measurement Ontology"	
INVESTIGATION
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INVESTIGATION PUBLICATIONS
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INVESTIGATION CONTACTS
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STUDY
Study Identifier	"E-GEOD-7584"
Study Title	"Transcription profiling of rat intestinal epithelial IEC-6 cells treated with non-steroidal anti-inflammatory agents to investigate gastrointestinal toxicity of NSAIDs"
Study Description	"IEC-6 cells were incubated with indomethacin, NS-398, or SC-560 for 72 hours. [original description: Non-steroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well-documented gastrointestinal (GI) toxicity. Presently, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial; cells (IEC-6) were treated with NSAIDs that either [inhibit] (indomethacin, NS-398) or lack (SC-560) inhibitory effects on intestinal epithelial cell migration. Bioinformatic analysis of array data suggested that NSAIDs with adverse GI effects either decrease the gene expression of the calpains or increase the gene expression of the endogenous calpain inhibitor calpastatin. Calpains have been shown previously to modulate the migration of a variety of cells in different physiological contexts. Our experimental results suggest that the altered expression of calpain genes may contribute to the adverse effects of NSAIDs on intestinal; epithelial restitution. Microarray analysis has generated the novel hypothesis that the GI toxicity of NSAIDs may be attributed in part to drug-induced changes in the expression and activity of calpains. Experiment Overall Design: Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398) or lack (SC-560) inhibitory effects on intestinal epithelial cell migration. Samples were then pooled to obtain sufficient material for gene array analysis. The pooled samples were used to hybridize 4 gene array chips for each biological sample.]"
Comment[Study Grant Number]	""
Comment[Study Funding Agency]	""
Study Submission Date	""
Study Public Release Date	"2009-03-27"
Study File Name	"s_E-GEOD-7584_study_samples.txt"
STUDY DESIGN DESCRIPTORS
Study Design Type	"unknown_experiment_design_type"	"transcription profiling by array"
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STUDY PUBLICATIONS
Study PubMed ID	"18281595"
Study Publication DOI	"10.1124/jpet.107.127720"
Study Publication Author List	"Raveendran NN, Silver K, Freeman LC, Narvaez D, Weng K, Ganta S, Lillich JD."
Study Publication Title	"Drug-induced alterations to gene and protein expression in intestinal epithelial cell 6 cells suggest a role for calpains in the gastrointestinal toxicity of nonsteroidal anti-inflammatory agents."
Study Publication Status	"Published"
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STUDY FACTORS
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STUDY ASSAYS
Study Assay File Name	"a_E-GEOD-7584_GeneChip_assay.txt"
Study Assay Measurement Type	"transcription profiling"
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Study Assay Technology Type	"DNA microarray"
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STUDY PROTOCOLS
Study Protocol Name	"P-G7584-5"	"P-G7584-1"	"P-G7584-2"	"P-G7584-7"	"P-G7584-8"	"P-G7584-9"	"P-G7584-4"	"P-G7584-3"	"P-G7584-6"
Study Protocol Type	"same as # 1"	"grow"	"specified_biomaterial_action"	"specified_biomaterial_action"	"specified_biomaterial_action"	"specified_biomaterial_action"	"nucleic_acid_extraction"	"labeling"	"labeling"
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Study Protocol Description	"Incubated for 72 hours at 37C in 5% CO2"	"Incubated for 72 hours at 37°C in 5% CO2."	"Vehicle Control with DMSO for 72 hours"	"Vehicle Control with 100µM Indomethacin for 72 hours"	"Vehicle Control with 100µM NS-398 for 72 hours"	"Vehicle Control with 1µM SC-560 for 72 hours"	"Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA)."	"Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C holdthermal cycler with a heated lid for incubations in this step. Use the 4 C 6 minutesfor the addition of reagents. 70 C hold4 C 6042 minutes C 10 minutes70 C hold4 a. Mix 1ul of total RNA (50ng/ul) C in thermalwith 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. C for 2 min.Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mix using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul 0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes.e. Incubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II.at 70 g. Spin briefly and cool C.sample to 4 Step 2. First cycle, second strand cDNA synthesis Note: Use C hold forthermal cycler without heated lid for incubations in this step. Use 4 the addition of reagents. C 120 minutes16 C hold4 C 1016 minutes C hold4 a Prepare the SS-Premix-1 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Mix by pipetting and spin briefly. d. C for 2 hours.Incubate the samples at 16 e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. Cf. Continue incubation at 16 for 10 minutes. Step 3. First cycle, double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate the double-stranded cDNA C for 2 hours.at -20 C.e. Centrifuge at 14,000rpm for 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at 14,000rpm for 5 C.minutes at 4 h. Remove ethanol and dry pellet in speed vacuum for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every 30 minutes.c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes to denature cRNA.b. Incubate at 70 c. Cool sample on ice for 2 minutes. d. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by pipetting. Spin briefly. C for 1 hour. Spin briefly.f. Incubate the sample at 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at C for 20 minutes.37 C for 5 minutes to inactivateh. Heat the sample at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold to add reagents.the 4 C 6 minutes70 C hold4 C 12016 minutes C hold4 C 10 minutes16 C hold4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 minutes.c. Incubate sample at 70 d. Cool the C and spin briefly.sample to 4 e. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours.g. Incubate at 16 h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10i. Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 minutes.Precipitate the double-stranded cDNA at -20 d. Centrifuge C.the sample at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the cDNA pellet by adding 800ul of 70% cold ethanol. f. C.Centrifuge the tube at 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a speed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step.at –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially after adding water, ensuring the cDNA pellet is completely dissolved. C for2. Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation. Step"	"Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation."
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STUDY CONTACTS
Study Person Last Name	"Raveendran"
Study Person First Name	"Nithya"
Study Person Mid Initials	""
Study Person Email	"nraveend@vet.ksu.edu"
Study Person Phone	""
Study Person Fax	""
Study Person Address	"Cellular Biophysics lab, Anatomy & Physiology, Kansas State University, 1600 Denison Avenue, Manhattan, 66506, USA"
Study Person Affiliation	"Kansas State University"
Study Person Roles	"submitter"
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