Investigation Title	Transcription profiling of Arabidopsis mutants lacking the K+ channels, akt1, cngc1 and cngc4	
Comment[Submitted Name]	Hampton: Differential gene expression patterns in Arabidopsis mutants lacking the K+ channels, akt1, cngc1 and cngc4.	
Experimental Design	strain_or_line_design	transcription profiling by array	
Experimental Design Term Source REF	mo	EFO	
Comment[ArrayExpressReleaseDate]		
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-NASC-31	
Comment[MAGETAB TimeStamp_Version]	2010-08-11 19:17:39 Last Changed Rev: 13058	
Experimental Factor Name	strain_or_line	
Experimental Factor Type	strain_or_line	
Experimental Factor Term Source REF	
Person Last Name	unknown	Hampton	
Person First Name	unknown	Corrina	
Person Mid Initials	
Person Email		corrina.hampton@hri.ac.uk	
Person Phone	+44 (0)115 951 3091	01789 470382	
Person Fax	+44 (0)115 951 3297	01789 470552	
Person Address	Plant Science Division, School of Biosciences, University of Nottingham, Sutton Bonnington Campus, Loughborough LE12 5RD, UK	Horticulture Research International Wellesbourne Warwick  CV35 9EF UK 	
Person Affiliation		Horticulture Research International	
Person Roles		biosource_provider;investigator;submitter	
Person Roles Term Source REF		mo	
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Experiment Description	Background: Release of the caesium radioisotope 137Cs during weapons testing and industrial activity has contaminated thousands of hectares of agricultural land. Ingesting 137Cs has damaging and, sometimes, fatal effects.  Most Cs enters the food chain through plants. The generation of _safe_ crops that exclude Cs and can be cultivated on contaminated land requires knowledge about the mechanisms for Cs uptake. Caesium is chemically similar to potassium (K) and might enter plants through K+ transporters in the plasma membrane of root cells.  To determine which transporters mediate Cs entry to plants, we have compared the accumulation of Cs and K by wildtype Arabidopsis with mutants lacking specific K+ transporters. Preliminary results showed that Cs concentration in the shoots of akt1-1, cngc1 and cngc4 (obtained from the Wisconsin T-DNA knockout facility) differed significantly from the Wassilewskija wildtype (Ws-2). A cursory investigation of their transcriptome, using the Affymetrix Arabidopsis 8K GeneChip, showed that the expression of several genes encoding K+ transporters differed between mutants and wildtype plants.  The aim of this GarNet project is to confirm the previous observations and to identify further genes that are differentially expressed in mutant and wildtype plants and which might impact on Cs accumulation.  Methods: Arabidopsis mutants akt1-1 (N3762), cngc1 and cngc4 and their parental ecotype Wassilewskija -2 (N1601) will be sown on MS agar and transferred to hydroponics 21 days after germination. Seedlings will be grown for a further 7 days on full nutrient solution under continuous light in a Saxcil growth cabinet. RNA will be extracted from roots of mutant and parent (control) plants at the same growth stage and twelve complete-genome Affymetrix GeneChips (3 biological replicates of material from wildtype and 3 mutants) are requested to determine the differences in their transcriptome under comparable environmental conditions.	
Protocol Name	NASCGrowProt:448	NASCExtractProtocol:13	NASCLabelProtocol:1	Affymetrix:Protocol:Hybridization-EukGE-WS2v4	Affymetrix:Protocol:Percentile	Affymetrix:Protocol:ExpressionStat	
Protocol Type		nucleic_acid_extraction	labeling	hybridization	feature_extraction	bioassay_data_transformation	
Protocol Description	Plants were grown in growth rooms at 80% humidity and a temperature of 22C. Stratification was carried out by imbibition for 3-5 days at 4C. Lighting regime was continuous light for 50-80 imol photons m-2 s-1. The medium used was 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (full-strength formulation Murashige and Skoog). After 18 days on MS agar plants were transferred to a hydroponic system containing full strength nutrient solution containing KH2PO4 0.25mM, KOH 0.50mM, MgSO4.7H2O 0.75mM, CaCl2.2H2O 0.03mM, FeNaEDTA 0.10mM, Ca(NO3)2.4H2O 4.00mM, H3BO3 30.0µM, MnSO4.4H2O 10.0µM, ZnSO4.7H2O 1.0µM, CuSO4.5H2O 3.0µM, Na2MoO4.2H2O 0.5µM. All plants were grown to growth stage (Boyes key) 3.90. Whole roots were harvested for RNA extraction. 20-40 plants were pooled per extract.	Protocol followed according to manufacturers instructions.	RNA samples were quality controlled using the Agilent 2100 Bioanalyzer. 100 pmol T7-(dT)24 primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3')  was hybridized with the Total RNA ( 8 - 12ug) at 70°C for 10 minutes.  First strand cDNA was synthesized by reverse transcription using 400 units SuperScript II Reverse Transcriptase in a reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.5 mM dNTPs at 42C for 1 hour.  The reaction was then put on ice for 2 mins, followed by second strand cDNA synthesis.  130ul of the second strand reaction mix was added to the 20ul  first strand synthesis reaction. The second strand reaction mix  contained 40 units E. coli DNA polymerase I, 10 units E. coli DNA ligase, 2 units  E. coli RNase H and 0.2 mM dNTPs in a buffer containing 20 mMTris-HCl (pH 6.9), 90 mM KCl, 4.6 mM MgCl2, 10mM (NH4)SO4 and 0.15 mM ß-NAD+. The reaction was incubated at 16 C for 2 hours.  10 units T4 DNA Polymerase was added and the reaction incubated for a further 5 minutes at 16 C, then the reaction was terminated using EDTA.  The double-stranded cDNA was cleaned up using the cDNA Cleanup Spin Column supplied in the Affymetrix GeneChip Sample Cleanup Module.  The double-stranded cDNA was used as a template for in Vitro transcription of biotin-labeled cRNA using the ENZO BioArray RNA Transcript Labeling Kit supplied by Affymetrix.  The Transcript Labeling kit produces large amounts of hybridizable biotin-labeled RNA targets by in Vitro transcription from bacteriophage T7 RNA polymerase promoters.  The reaction mixture containing Biotin-Labeled ribonucleotides (ATP, GTP, CTP, UTP with Bio-UTP and Bio-CTP), DTT, RNase inhibitor mix and T7 RNA polymerase was incubated at 37 C for 5 hrs, gently mixing every 30 minutes during the incubation. This produced an average of 40ug biotin-labeled cRNA . The biotin-labeled cRNA  was cleaned up using the cRNA Cleanup Spin Column supplied in the Affymetrix GeneChip Sample Cleanup Module.  The quantity and quality of the cRNA was measured using the Agilent 2100 Bioanalyzer. The total amount of cRNA was calculated, the starting quantity of RNA subtracted to give the quantity of adjusted cRNA.  The cRNA was fragmented by metal-induced hydrolysis to break down full-length cRNA to 35-200 base fragments.  15ug of adjusted cRNA was used to prepare 300ul of hybridization cocktail of which 200ul was hybridized with the GeneChip.	Title: Fluidics Station Protocol. Description: 	Title: Affymetrix CEL Analysis (Percentile). Description: 	Title: Affymetrix CHP Analysis (ExpressionStat). Description: 	
Protocol Parameters	location;Humidity;Temperature;	
Protocol Hardware	
Protocol Software				MicroArraySuite 5.0	MicroArraySuite 5.0	MicroArraySuite 5.0	
Protocol Contact	
Protocol Term Source REF		mo				The MGED Ontology	
SDRF File	E-NASC-31.sdrf.txt	
Term Source Name	nasc		ncbitax	ArrayExpress	mo	EFO	The MGED Ontology	
Term Source File	http://arabidopsis.info/catalogue.html		http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
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