Investigation Title	Transcription profiling of wild type and jasmonate deficient mutant aos Arabidopsis plants in control conditions	
Comment[Submitted Name]	FARMER-Arabidopsis Jasmonate Deficient	
Experimental Design	genetic_modification_design	transcription profiling by array	
Experimental Design Term Source REF	mo	EFO	
Comment[ArrayExpressReleaseDate]	2008-02-01	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-ATMX-36	
Comment[MAGETAB TimeStamp_Version]	2011-06-28 11:35:50 Last Changed Rev: 14857	
Experimental Factor Name	genotype	
Experimental Factor Type	genotype	
Experimental Factor Term Source REF	
Person Last Name	Farmer	
Person First Name	Edward	
Person Mid Initials	E	
Person Email	edward.farmer@unil.ch	
Person Phone	0041 21 692 4228	
Person Fax	0041 21 692 4195	
Person Address	Bâtiment BIOPHORE, Lausanne, campus Dorigny, 1015, Switzerland	
Person Affiliation	Departement de Biologie Moleculaire Végétale	
Person Roles	submitter	
Person Roles Term Source REF		
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	2008-02-01	
PubMed ID	18996838	
Publication DOI	18996838	
Publication Author List	Mene-Saffrane, Laurent; Dubugnon, Lucie; Chetelat, Aurore; Stolz, Stephanie; Gouhier-Darimont, Caroline; Farmer, Edward E.	
Publication Title	Non-enzymatic oxidation of trienoic fatty acids contributes to reactive oxygen species management in Arabidopsis	
Publication Status	journal_article	
Publication Status Term Source REF	The MGED Ontology	
Experiment Description	comparison of the transcriptome of Columbia WT and the jasmonate deficient mutant aos (allene oxide synthase)in control conditions.	
Protocol Name	P-CAGE-25009	P-CAGE-25010	P-CAGE-25015	P-CAGE-25011	P-CAGE-25014	P-CAGE-25013	
Protocol Type	nucleic_acid_extraction	labeling	labeling	hybridization	image_acquisition	bioassay_data_transformation	
Protocol Description	1.Total RNA extraction<br> Grind 3-6g (about 12 plants) of Arabidopsis leaves in liquid N2 with mortar and pestle. Transfer to 50ml Falcon tube.<br> <br> Add 18ml of 1:2:1 solution (precipitation at RT)<br> o 1 volume of 2M Tris-HCl pH 8.2<br> o 2 volumes of 0.5M EDTA pH 8.0 (DEPC treated)<br> o 1 volume of 20% SDS<br> <br> Add 9 ml of phenol saturated with 0.1M Tris-HCl pH 8.2. Stir for minimum 15min.<br> Add 9ml chloroform. Stir for minimum 10min.<br> Centrifuge 3600 x g 15min.<br> Treat upper phase (aqueous) twice with 9ml chloroform, centrifuging 3600 x g 15min.<br> Transfer 15ml of aqueous phase to 30ml disposable polypropylene tube and add equal volume of ice-cold 6M LiCl (DEPC treated). Precipitate O/N on ice at 4°C.<br> Centrifuge 9800 x g 20min 4°C.<br> Resuspend into 2.5ml H2O, add 0.25ml 3M NaAcetate pH 5.2 and 3 volumes (8.3ml) of EtOH. Precipitate at RT for ca 60min.<br> Centrifuge 9800 x g 20min 16°C, rinse with 5ml 70% EtOH, centrifuge 9800 x g 5min, air dry pellet 15min and resuspend in 0.5 ml H2O.<br> Quantify RNA using Nanodrop. Dilute 2µl in 400µl 10 mM Tris pH 8.<br>  <br> 2.Total RNA purification-Use Rneasy Mini Kit (QIAGEN) <br> mix thoroughly 150 µg total RNA with 350 µl Buffer RLT/1% beta-Mercaptoethanol<br> Add 250 µl ethanol 100% to each sample, mix by pipetting<br> Apply to an Rneasy mini column, centrifuge 8000 x g 15s, discard the flowthrough<br> Wash the column with 500 µl Buffer RPE, centrifuge 8000 x g 15s, discard the flowthrough<br> Add another 500 µl RPE buffer, centrifuge 8000 x g 2 min to dry the silica-gel membrane<br> Transfer the column into a new 2 ml collection tube and centrifuge 8000 x g 1 min to eliminate any Buffer RPE carryover<br> Elution : transfer the column into a 1.5 ml collection tube, add 40 µl H2O nuclease free 50°C preheated, centrifuge 8000 x g 1 min (twice)<br> Measure concentration (0.5 µl in 200 µl 10 mM Tris pH8)<br> 	Concentrate RNA using microcon.<br> Take 100-150 µg RNA in 80 µl H2O, put in microcon column. Centrifuge 4min at 14000g. Turn column in clean tube. Centrifuge for 3 min at 1000 g. Measure concentration (0.5 µl in 300 µl 10 mM Tris pH8)<br> <br> Reverse transcription.<br> Prepare two 200 µl PCR tubes according to the recipe below:<br> 100 µg of total RNA + 2 µl oligo dT21 mer (1 mg/ml) + enough water to achieve 13.4 µl final volume. Heat 5 min at 70oC in PCR machine. Leave at RT for 5 min. Prepare mix according to the following recipe: <br> 6 µl 5x SuperScript II Buffer +<br> 3 µl 0.1 M DTT +<br> 0.6 µl dNTPS (25 mM dATP, dGTP, dTTP; 10 mM dCTP) +<br> 2 µl SuperScript II Reverse Transcriptase (Invitrogen)+<br> 2 µl RNase Inhibitor (Invitrogen).<br> Add 13.6 µl mix per tube. <br> Add: 3 µl/tube Cy3-dCTP (1 mM) for each sample. Mix well and incubate at 42oC for 2 hr in PCR machine.<br> <br> End of probe preparation.<br> Pool the two tubes together (corresponding Cy3 and Cy5 samples).<br> Add: 2.65 µl 25 mM EDTA + 3.30  µl 1 M NaOH.<br> Incubate 10 min at 65oC in PCR machine.<br> Add: 3.3 µl 1 M HCl + 5 µl 1 M Tris-HCl, pH 6.8.<br> Purify probe with Qiagen MinElute PCR purification kit (#28004)<br> Mix 5 volumes (370 µl) buffer PB with 1 volume of probe (74 µl). <br> Add 3 µl 3M NaAc. pH 5.2 and mix. Transfer to column.<br> Spin 1 min at &#8805; 10,000 x g.  Discard flowthrough.<br> Add 750 µl buffer PE to column. Spin 1 min at &#8805; 10,000 x g.  Discard flowthrough. Spin 1 additional min. Transfer column to new 1.5 mL tube.  Add 10 ml of buffer EB to the center of the membrane.  Wait 1 min then centrifuge for 1 min. Repeat the elution step  : ~20 ml eluate. Quantify using Nanodrop (2 µl/20).<br> Mix in a a 200 µl PCR tube: <br> labeled probe 18 µl +<br> H2O 53.2 µl +<br> SSC 20X 15 µl +<br> SDS 10% 3.8 µl +<br> tRNA 2 µg/µl 10 µl +<br> Total 100 µl.<br> 	Concentrate RNA using microcon. Take 100-150 µg RNA in 80 µl H2O, put in microcon column. Centrifuge 4min at 14000g. Turn column in clean tube. Centrifuge for 3 min at 1000 g. Measure concentration (0.5 µl in 300 µl 10 mM Tris pH8) Reverse transcription. Prepare two 200 µl PCR tubes according to the recipe below: 100 µg of total RNA + 2 µl oligo dT21 mer (1 mg/ml) + enough water to achieve 13.4 µl final volume. Heat 5 min at 70oC in PCR machine. Leave at RT for 5 min. Prepare mix according to the following recipe: 6 µl 5x SuperScript II Buffer + 3 µl 0.1 M DTT + 0.6 µl dNTPS (25 mM dATP, dGTP, dTTP; 10 mM dCTP) + 2 µl SuperScript II Reverse Transcriptase (Invitrogen)+ 2 µl RNase Inhibitor (Invitrogen). Add 13.6 µl mix per tube. Add: 3 µl/tube Cy5-dCTP (1 mM) for each sample. Mix well and incubate at 42oC for 2 hr in PCR machine. End of probe preparation. Pool the two tubes together (corresponding Cy3 and Cy5 samples). Add: 2.65 µl 25 mM EDTA + 3.30 µl 1 M NaOH. Incubate 10 min at 65oC in PCR machine. Add: 3.3 µl 1 M HCl + 5 µl 1 M Tris-HCl, pH 6.8. Purify probe with Qiagen MinElute PCR purification kit (#28004) Mix 5 volumes (370 µl) buffer PB with 1 volume of probe (74 µl). Add 3 µl 3M NaAc. pH 5.2 and mix. Transfer to column. Spin 1 min at &#8805; 10,000 x g. Discard flowthrough. Add 750 µl buffer PE to column. Spin 1 min at &#8805; 10,000 x g. Discard flowthrough. Spin 1 additional min. Transfer column to new 1.5 mL tube. Add 10 ml of buffer EB to the center of the membrane. Wait 1 min then centrifuge for 1 min. Repeat the elution step : ~20 ml eluate. Quantify using Nanodrop (2 µl/20). Mix in a a 200 µl PCR tube: labeled probe 18 µl + H2O 53.2 µl + SSC 20X 15 µl + SDS 10% 3.8 µl + tRNA 2 µg/µl 10 µl + Total 100 µl. 	Prepare slide with coverslip (Lifterslip 25x60mm, Erie Scientific) in chamber with 13 microl 3x SSC per groove. Heat probe 1 min at 95oC. Spin 1 min at high speed. Rapidly add probe to slide. Close chamber and immerge in 64oC bath. Hybridize overnight.  	Slides are scanned at variable laser power and PMT voltage so that the number of saturated spots is below 1% and so that spots in the Cy3 and Cy5 channels have the same average intensity.	The average fluorescence intensity for each fluor and for each gene   was determined using the ImaGene program (BioDiscovery Inc., Los   Angeles, CA). Median background fluorescence around each gene spot was calculated and subtracted from each spot. Signal values <500 (two   to three times the average background stdev) were manually raised to   500 to avoid extreme expression ratios. Subgrid Loess normalization   was performed using the limma library of the R statistical software (Yang YH, Dutoit S, Luu P, Lin DM, Peng V, Ngai J, Speed T: Normalization for cDNA microaaray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acid Res. 2002, 30(4):e15).  	
Protocol Parameters	Amplification;Extracted product;	Amount of nucleic acid labeled;Used Label;Amplification;	Amount of nucleic acid labeled;Amplification;Used Label;	Temperature;Volume;Time;Quantity of label target used;Chamber type;	
Protocol Hardware					ScanArray 4000 [PerkinElmer]	
Protocol Software					ScanArray Express [PerkinElmer]	
Protocol Contact	
Protocol Term Source REF		mo	mo	
SDRF File	E-ATMX-36.sdrf.txt	
Term Source Name	EFO		The MGED Ontology	abrc_stock	mo	EFO	The MGED Ontology	
Term Source File	http://www.ebi.ac.uk/efo/		http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.arabidopsis.org/servlets/Search?action=new_search&type=germplasm	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version