Investigation Title	Transcription profiling of human tamoxifen treated myeloid leukemia K562-MERT cells	
Comment[Submitted Name]	gene expression in K562-MERT cells	
Experimental Design	transcript_identification_design	transcription profiling by array	
Experimental Design Term Source REF		EFO	
Comment[ArrayExpressReleaseDate]	2004-06-30	
Comment[AEMIAMESCORE]	2	
Comment[ArrayExpressAccession]	E-MEXP-91	
Comment[MAGETAB TimeStamp_Version]	2011-08-18 14:33:38 Last Changed Rev: 14857	
Experimental Factor Name	disease_state	
Experimental Factor Type	disease_state	
Experimental Factor Term Source REF	
Person Last Name	Shetzline	
Person First Name	Susan	
Person Mid Initials	E	
Person Email	shetzlin@mail.med.upenn.edu	
Person Phone	215.898.5101	
Person Fax	215.573.7049	
Person Address	421 Curie Blvd. BRB II/III Rm. 727	
Person Affiliation	Medicine	
Person Roles	submitter	
Person Roles Term Source REF	mo	
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2004-06-30	
PubMed ID	15187020	
Publication DOI	15187020	
Publication Author List	
Publication Title	
Publication Status	journal_article	
Publication Status Term Source REF	mo	
Experiment Description	K562 cells, a myeloid leukemia cells line, were engineered to express a tamoxifen inducible dominant negative Myb (MERT).  K562-MERT cells were cultured for 3 days in the absence and presence of 1 uM tamoxifen.  The RNA was then extracted from the untreated and tamoxifen treated K562-MERT cells and submitted to Incyte Genomics for poly(A) RNA selection, probe preparation, hybridization of the labeled cDNA to the micorarray chip (Incyte Genomics) and data analysis. 	
Protocol Name	P-MEXP-2627	P-MEXP-3046	P-MEXP-2628	P-MEXP-2629	P-MEXP-3047	P-MEXP-2630	P-MEXP-2759	P-MEXP-3612	
Protocol Type	grow	specified_biomaterial_action	specified_biomaterial_action	nucleic_acid_extraction	labeling	labeling	hybridization	bioassay_data_transformation	
Protocol Description	K562 cells engineered to express a tamoxifen inducible dominant negative Myb (MERT) were maintainined at 37 degrees C, 5% C02 in RPMI supplemented with 10% FBS, 1% L-glutamine, and 0.5% penicillin/steptomycin.	K562 cells engineered to express tamoxifen inducible dominant negative Myb (MERT) were cultured in RPMI supplemented with 10% FBS, 1% L-glutamine, and 0.5% penicillin/streptomycin for three days.	K562 cells engineered to express tamoxifen inducible dominant negative Myb (MERT) were cultured in RPMI supplemented with 10% FBS, 1% L-glutamine, and 0.5% penicillin/streptomycin for three days in the absence and presence of 1 uM tamoxifen.	Following a three day culture of K562-MERT cells in the absence and presence of 1 uM tamoxifen, total RNA was extracted using RNeasy Mini kit (Qiagen) according to the manufacturer's specifications.  The RNA was submitted to Incyte for polyA RNA selection using Oligotex mRNA isolation kit (Qiagen).  Using a T7-based amplification kit (Incyte Genomics), the mRNA was subsequently subjected to linear amplification.  	At Incyte, amplified cRNA and unamplified mRNA from untreated K562-MERT cells were purified using a Agilent 2100 Bioanalyzer (Agilent Technologies) and labeled with Cy3 dye using a custom labeling kit (Incyte Genomics).  In brief, the reverse transcription labeling reactions were perfomed in 50 mM Tris-Cl pH 8.3, 75 mM KCl, 15 mM MgCl2, and 4 mM DTT.  Each reaction contained 200 ng of purified cRNA or mRNA, 2 ug Cy3 random 9-mer (Trilink), 2 mM dNTPs (0.5 mM each), 20 U RNase inhibitor (Ambion), and 200 U MMLV RNase H-free reverse transcriptase (Promega).  Cy3 cDNA products were purified using a size exclusion column, concentrated by ethanol precipitation, and resuspended in hybridization buffer.	At Incyte, amplified cRNA and unamplified mRNA from tamoxifen treated K562-MERT cells were purified using a Agilent 2100 Bioanalyzer (Agilent Technologies) and labeled with Cy5 dye using a custom labeling kit (Incyte Genomics).  In brief, the reverse transcription labeling reactions were perfomed in 50 mM Tris-Cl pH 8.3, 75 mM KCl, 15 mM MgCl2, and 4 mM DTT.  Each reaction contained 200 ng of purified cRNA or mRNA, 2 ug Cy5 random 9-mer (Trilink), 2 mM dNTPs (0.5 mM each), 20 U RNase inhibitor (Ambion), and 200 U MMLV RNase H-free reverse transcriptase (Promega).  Cy5 cDNA products were purified using a size exclusion column, concentrated by ethanol precipitation, and resuspended in hybridization buffer.	Labeled cDNA probes from untreated and treated K562-MERT cells were hybridized to a micrarray chip containing 10,000 PCR products.  In a microarray hybridization chamber (Incyte Genomics), 200 ng of Cy3 and Cy5-labeled cDNA probes were resuspended in 5X SSC, 0.2% SDS, 1 mM DTT, applied to the microarray chip under a glass cover slip, and incubated for 6 hours at 60 degrees C.  The microarray chip was then washed in 1X SSC/0.2% SDS for 10 min at 45 degrees C followed by a 3 min wash at room temperature in 0.1X SSC/0.2%.  The washed microarray chip was dried by centrifugation.  	GEMTools image analysis software (Incyte Genomics) uses an algorithm to quantify the signal and background intensity for each feature on the microarray chip. Using a signal-to-background threshold of 2.5, GEMTools derives the corrected signal intensity at each feature.  The ratio of the Cy3 signal to the balanced Cy5 signal yields the balanced differential expression or fold-change in each gene's expression on the microarray chip.  When the ratio of the Cy3 signal to balanced Cy5 signal is less than 1, the ratio of the balanced Cy5 signal to the Cy3 signal is calculated instead and assigned a negative number.  Therefore, the positive numbers in the balanced DiffExpr column in our microarray data set denote the genes that are decreased when endogenous Myb activity is abrogated by our conditionally active dominant negative Myb.  The negative numbers in the balanced DiffExpr column indicate those genes that are increased when endogenous Myb activity is inhibited by dominant negative Myb.    	
Protocol Parameters	media;min temperature;start time;			Extracted product;Amplification;	Amplification;Label used;Amount of nucleic acid labeled;	Amplification;Amount of nucleic acid labeled;Label used;	temperature;Chamber type;Quantity of label target used;time;Volume;	
Protocol Hardware	
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SDRF File	E-MEXP-91.sdrf.txt	
Term Source Name	mo		ArrayExpress	EFO	mo	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ebi.ac.uk/arrayexpress	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version