Comment[ArrayExpressAccession]	E-GEOD-35174
MAGE-TAB Version	1.1													
Public Release Date	2015-01-18
Investigation Title	Development of miRNA expression signatures of MEFs deficient in a ER stress mediator XBP1 for tunicamycin treatment													
Comment[Submitted Name]	Development of miRNA expression signatures of MEFs deficient in a ER stress mediator XBP1 for tunicamycin treatment
Experiment Description	To further development of our miRNA expression approach to ER stress, we have employed miRNA microarray expression profiling as a discovery platform to identify ER stress-responsible ones.   Mouse embryonic fibroblasts (MEFs) deficient in a ER stress mediator XBP1 were treated with tunicamycin for 12 or 24 hrs. miRNAs responsible for tunicamycin-treatment for 12hrs in XBP1-dependent manner were extracted. Among them, expression of three miRNAs (miR-23a, miR-27a, miR-24-2) was quantified in the RNA samples from  the same as the microarray, and COS7 cells by real-time PCR, confirming existence of similar mechanisms of trancriptional repression in ER stress by tunicamycin treatment. Mouse embryonic fibroblasts (MEFs) deficient in a ER stress mediator XBP1 were treated with 2ug/mL tunicamycin for 12 or 24 hrs. Two independent experiments were performed at each time (untreated, 12 or 24 hrs). miRNAs responsible for tunicamycin-treatment for 12hrs in XBP1-dependent manner were extracted.													
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl												
Person Last Name	Oyadomari	Ito	Mori	Zheng	Miyamoto	Tsugawa	Kurahashi	Funahashi	Sato	Miura	Takahara	Oyadomari	Miyake	Oyadomari
Person First Name	Seiichi	Taiji	Tomoko	Robert	Chinobu	Kazue	Kiyoe	Mari	Ryosuke	Naoko	Kazuna	Miho	Masato	Seiichi
Person Email	oyadomar@genome.tokushima-u.ac.jp													
Person Affiliation	Institute for Genome Research													
Person Phone	81-88-633-9450													
Person Address	Division of Molecular Biology, Institute for Genome Research, 3-18-15 Kuramoto, Tokushima, Tokushima, Japan													
Person Roles	submitter													
Protocol Name	P-GSE35174-1	P-GSE35174-5	P-GSE35174-6	P-GSE35174-2	P-GSE35174-3	P-GSE35174-4	P-GSE35174-7							
Protocol Description	The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol miRNA_107_Sep09 and Grid: 021828_D_20081121) to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. ID_REF =  VALUE = Normalized signal intensity (log2)	Directly Cyanine-3 (Cy3)-labeled miRNA was prepared from 100 ng total RNA using the miRNA Complete Labeling and Hyb kit (Agilent) according to the manufacturer's instructions, followed by MicroBioSpin 6 Columns purification (BioRad, Hercules, CA) and evaporation to dryness.	Dried Cy3-labelled miRNA was resuspended in a reaction volume of 45 ul containing 45000000 fold-diluted Agilent Hyb Spike-In, 1x Agilent Gene Expression Blocking Agent and 1x Agilent Hi-RPM Hybridization Buffer following the manufacturers instructions. After denaturing the mixture by heating at 100 ºC for 5 minute and immediate cooling on ice for 5 minute, the mixture was hybridized to Agilent Mouse miRNA Microarrays (G4472B) for 21.5 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute with 37°C GE Wash buffer 2 (Agilent), then air-dried immediately.	MEFs were treated with DMEM supplemented with 10% fetal bovine serum, 55uM 2-mercaptoethanol, 1x MEM non-essential amino acids, 100units/ml penicillin and 100ug/ml streptomycin containing 2ug/mL tunicamycin for 12 or 24 hours at 37 ºC in a humidified incubator with 5% CO2.	MEFs were cultured in DMEM supplemented with 10% fetal bovine serum, 55uM 2-mercaptoethanol, 1x MEM non-essential amino acids, 100units/ml penicillin and 100ug/ml streptomycin at 37 ºC in a humidified incubator with 5% CO2.	total RNA was prepared using the mirVana miRNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop spectrophotometer and quality was monitored by agarose gel electrophoresis of RNA under denaturing conditions.	Slides were scanned immediately after washing and drying on the Agilent DNA Microarray Scanner (G2505C) using miRNA scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%, NoXDR setting).							
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol							
Experimental Factor Name	treatment	time	genotype											
Experimental Factor Type	treatment	time	genotype											
Comment[SecondaryAccession]	GSE35174													
Comment[GEOReleaseDate]	2015-01-18													
Comment[ArrayExpressSubmissionDate]	2012-01-18													
Comment[GEOLastUpdateDate]	2015-01-18													
Comment[AEExperimentType]	transcription profiling by array													
SDRF File	E-GEOD-35174.sdrf.txt