Investigation Title	Transcription profiling of activated and tolerant CD4+ T cells of Tg4 TCR transgenic mice to discover differentially expressed genes with the aim of gaining new insights about the molecular and genetic basis underlying autoimmune disease	
Comment[Submitted Name]	Repetitive antigen stimulation changes the genetic program of activated T cells and results in antigen specific peripheral T cell tolerance	
Experimental Design	dose_response_design	transcription profiling by array	
Experimental Design Term Source REF		EFO	
Comment[ArrayExpressReleaseDate]	2005-03-31	
Comment[AEMIAMESCORE]	5	
Comment[ArrayExpressAccession]	E-MEXP-283	
Comment[MAGETAB TimeStamp_Version]	2011-06-28 19:46:35 Last Changed Rev: 14857	
Experimental Factor Name	Ac1-9 peptide treatment	Number of doses of Ac1-9 in vivo	
Experimental Factor Type	compound_treatment_design	dose	
Experimental Factor Term Source REF	
Person Last Name	khalid	
Person First Name	sabah	
Person Mid Initials	
Person Email	sabah.khalid@brunel.ac.uk	
Person Phone	01895 274000	
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Person Address	Brunel University, Uxbridge, Middlesex, UK	
Person Affiliation	Bioinformatics	
Person Roles	submitter	
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Normalization Type	
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Date of Experiment	
Public Release Date	2005-03-31	
PubMed ID	
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Publication Status Term Source REF	
Experiment Description	Our experiments examine gene expression profiles from activated and tolerant CD4+ T cells of Tg4 TCR transgenic mice to discover differentially expressed genes with the aim of gaining new insights about the molecular and genetic basis underlying autoimmune disease.	
Protocol Name	P-MEXP-7954	P-MEXP-7941	P-MEXP-7937	P-MEXP-3950	P-MEXP-3951	P-MEXP-3953	P-MEXP-3955	
Protocol Type	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	nucleic_acid_extraction	labeling	hybridization	bioassay_data_transformation	
Protocol Description	The origin of the biological sample Female Tg4 TCR transgenic mice expressing the abTCR (Va4, Vb8.2) of the Ac1-9 specific T cell hybridoma 1934.4 derived from an encephalitogenetic T cell clone, were bred onto the B10.PL (H-2u) background and maintained under standard conditions at room temperature at the School of Medical Sciences, Bristol and were between 6 and 12 weeks of age when used for experiments. Manipulation of biological samples and protocols used The screening of TCR transgenic mice was performed using 2-colour immunofluorescent staining of peripheral blood lymphocytes with anti-CD4 and anti-Vb8, monoclonal antibody. The spleens from Tg4 TCR transgenic mice were used as a source of CD4+ T cells. The wild type, acetylated, N-terminal peptide of murine MBP (Ac1-9 (4K) AcASQKRPRSQR) was modified to have a tyrosine substituting the wild-type lysine in position 4 (Ac1-9 (4Y), using standard Fmoc chemistry on an AMS 422 multiple peptide synthesiser (Abimed, Langenfield, Germany). The modified Ac1-9 [4Y] peptide (derived from myelin basic protein (MBP)) was solubilised in phosphate-buffered saline (PBS) at 4mg/ml and 10 doses of 25ml administered intranasally under light halothane anaesthesia at 3-4 day intervals. CD4+ T cells were purified from Tg4 mice after 10 intranasal peptide administrations. For in vitro stimulation, cell suspensions were prepared from mice spleens immediately after the 10th intranasal administration and seeded at 1.5x106 cells/ml in RPMI (5% FCS). Cells were cultured  without the peptide Ac1-9 and with IL-2 (20U/ml; R and D Systems) for three days. CD4+ T cells were obtained by positive selection using magnetic beads coated with anti-CD4 monoclonal antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany) according to the manufactures instructions.	The origin of the biological sample Female Tg4 TCR transgenic mice expressing the abTCR (Va4, Vb8.2) of the Ac1-9 specific T cell hybridoma 1934.4 derived from an encephalitogenetic T cell clone, were bred onto the B10.PL (H-2u) background and maintained under standard conditions at room temperature at the School of Medical Sciences, Bristol and were between 6 and 12 weeks of age when used for experiments. Manipulation of biological samples and protocols used The screening of TCR transgenic mice was performed using 2-colour immunofluorescent staining of peripheral blood lymphocytes with anti-CD4 and anti-Vb8, monoclonal antibody. The spleens from Tg4 TCR transgenic mice were used as a source of CD4+ T cells. The wild type, acetylated, N-terminal peptide of murine MBP (Ac1-9 (4K) AcASQKRPRSQR) was modified to have a tyrosine substituting the wild-type lysine in position 4 (Ac1-9 (4Y), using standard Fmoc chemistry on an AMS 422 multiple peptide synthesiser (Abimed, Langenfield, Germany). The modified Ac1-9 [4Y] peptide (derived from myelin basic protein (MBP)) was solubilised in phosphate-buffered saline (PBS) at 4mg/ml and 10 doses of 25ml administered intranasally under light halothane anaesthesia at 3-4 day intervals. CD4+ T cells were purified from Tg4 mice after the 10th intranasal peptide administration and stimulated in vitro.  Cell suspensions were prepared from the mice spleens immediately after the 10th intranasal administration and seeded at 1.5x106 cells/ml in RPMI (5% FCS). Cells were cultured with Ac1-9 (1mg/ml) and IL-2 (20U/ml; R and D Systems) for three days. CD4+ T cells were obtained by positive selection using magnetic beads coated with anti-CD4 monoclonal antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany) according to the manufactures instructions.  	The origin of the biological sample Female Tg4 TCR transgenic mice expressing the abTCR (Va4, Vb8.2) of the Ac1-9 specific T cell hybridoma 1934.4 derived from an encephalitogenetic T cell clone, were bred onto the B10.PL (H-2u) background and maintained under standard conditions at room temperature at the School of Medical Sciences, Bristol and were between 6 and 12 weeks of age when used for experiments. Manipulation of biological samples and protocols used The screening of TCR transgenic mice was performed using 2-colour immunofluorescent staining of peripheral blood lymphocytes with anti-CD4 and anti-Vb8, monoclonal antibody. The spleens from Tg4 TCR transgenic mice were used as a source of CD4+ T cells. The wild type, acetylated, N-terminal peptide of murine MBP (Ac1-9 (4K) AcASQKRPRSQR) was modified to have a tyrosine substituting the wild-type lysine in position 4 (Ac1-9 (4Y), using standard Fmoc chemistry on an AMS 422 multiple peptide synthesiser (Abimed, Langenfield, Germany). The modified Ac1-9 [4Y] peptide (derived from myelin basic protein (MBP)) was solubilised in phosphate-buffered saline (PBS) at 4mg/ml and 1 dose of 25ml administered intranasally under light halothane anaesthesia. CD4+ T cells were purified from naïve Tg4 mice 2 hours (A2) after a single intranasal peptide administration.	Protocol for preparing the hybridisation extract     Total RNA was extracted from purified CD4+ T cells from A0, A2, T0 and T2 spleens using TRIZOL reagent (GIBCO, Rockville, MD) according to the manufactures instructions.  If the starting material is tissue, homogenise in 1ml of TRIZOL reagent/50-100mg of tissue.  If the starting material is cells grown in suspension, lyse the cells in TRIZOL reagent by repetitive pipetting using     1ml/5-10x106 for animal, plant or yeast cells or per 1x107 for bacterial cells.   The first stage called phase separation involves incubating the homogenised samples for 5 minutes at 15-30degrees C, adding 0.2ml of chloroform/1ml of TRIZOL reagent, shaking the tubes vigorously by hand for 15 seconds, incubating them for 3 minutes at 15-30degrees C and finally centrifuging at a maximum of 12,000xg for 15 minutes at 2-8degrees C.   The second stage of RNA precipitation requires transferring the RNA containing aqueous phase to a fresh tube, adding 0.5ml of isopropyl alcohol per 1ml of TRIZOL reagent used for the initial homogenisation, incubating for 10 minutes at 15-30degrees C and finally centrifuging at a maximum of 12,000xg for 10 minutes at 2-8degrees C.    The final stage is the RNA wash.  The supernatant is removed and the RNA pellet washed once with 75% ethanol, adding 1ml of 75% ethanol per 1ml of TRIZOL reagent used for the initial homogenisation.  The sample is mixed by simple vortexing and centrifuged at no more than 7500xg for 5 minutes at 2-8degress C.  The RNA pellet is briefly air-dried and partially dissolved in Rnase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip and incubating for 10 minutes at 55-60degrees C.   	The protocol followed for the CyScribe Post-Labelling Kit, supplied by Amersham Biosciences, Amersham, UK, resulted in the production of Cy3 and Cy5 labelled cDNA probes.  30mg of total RNA from each sample was primed with 3ml of anchored oligo (dT) DNA primer and incubated at 70 degrees C for 5 minutes.  The RNA was reverse transcribed into cDNA using CyScript reverse transcriptase incorporating amino-allyl-dUTP (AA-dUTP).  The cDNA was purified using the manufactures instructions for the CyScribe GFX purification kit supplied by Amersham Biosciences, followed by the labelling of the amino-allyl modified cDNA using Cy3 and Cy5 dyes for dual colour microarray hybridisations.     Purified amino allyl modified cDNA from one synthesis reaction was dried down in a rotary evaporator until no liquid remained and was resuspended in 15ml of nuclease free water.  One aliquot of Cy3 or Cy5 dye was resuspended in 15ml of fresh 0.1M Sodium Bicarbonate pH 9.0 and immediately added to one tube of resuspended amino allyl modified cDNA and incubated at room temperature in the dark for one hour.  15ml 4M hydroxylamine was added to each coupling reaction, mixed by stirring and again incubated at room temperature in the dark, but only for 15 minutes.  The 30mg of total RNA obtained 4 times from the spleen cells of naïve mice was labelled with Cy3 dye and the 30mg obtained 4 times from the 4 samples was labelled with Cy5.     Labelled cDNA probes were purified using CyScribe GFXTM PCR purification columns according to the manufacturers instructions as provided in the CyScribe Post Labelling Kit supplied by Amersham Biosciences.  The Cy3 (30mg) and Cy5    (30mg) labelled cDNA (total of 60mg) were mixed for dual colour hybridisation, dried and resuspended in a microarray hybridisation buffer supplied in the CyScribe post labelling kit.   External controls (spikes)  The yield of labelled cDNA was determined by spiking the labelling reaction with 2ml of a 1:10 dilution in nuclease free water of [a-33P] dATP provided in the CyScribe Post Labelling Kit supplied by Amersham Biosciences followed by thin layer chromatography to quantify the amount of [a-33P] dATP incorporated as instructed by Amersham Biosciences protocol.  Quantification of the amount of incorporated [a-33P] dATP as a proportion of the total [a-33P] dATP in the reaction enables the calculation of cDNA yield.    	Microarray slides were pre-hybridised in 50% formamide, 5xSSC, 0.1%SDS and 1%BSA (supplied by Sigma-Aldrich, St.Lois, MO), washed in distilled water and dried.  The mixed purified labelled cDNA probes were dried and resuspended in a supplied microarray hybridisation buffer containing 50% formamide, 3 micrograms of oligonucleotide A40-60 and 3 micrograms of Cot-1 DNA.    This hybridisation mixture containing the labelled probes was pipetted onto the prepared microarray slide and covered with a cover slip.  Probe hybridisation was carried out at 42 degrees C for 14-18 hours overnight in a humid hybridisation chamber.  The arrays were washed at room temperature twice in 2xSSC for 5 minutes, twice in 0.1xSSC/0.1% SDS for 5 minutes and twice in 0.1xSSC for 5 minutes.  Arrays were immediately dried with a gentle air stream.  	Acuity Software - version 3.1.0.28, a microarray informatics platform for microarray data storage, data filtering and data analysis, again supplied by Axon Instruments was used for data analysis.  The spot quantitation matrices from GenePix were saved to Acuity for normalisation and data selection to produce final gene expression data matrices upon which conclusions could be reached.   Using Acuity, local background subtraction was performed and a ratio-based data normalisation algorithm was implemented to perform a constant linear normalisation normalising all spots equally.  In this case the data was normalised so that the mean of the ratio of medians of all features was equal to 1.  Log ratios of sample signal/reference signal were used for subsequent analysis with various statistical algorithms. 	
Protocol Parameters				Amplification;Extracted product;	Amplification;Amount of nucleic acid labeled;Label used;	Quantity of label target used;time;Chamber type;Volume;temperature;	
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Protocol Term Source REF				mo	mo	mo	mo	
SDRF File	E-MEXP-283.sdrf.txt	
Term Source Name	mo		ncbitax	ArrayExpress	EFO	mo	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://www.ebi.ac.uk/arrayexpress	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version