Investigation Title	Transcription profiling of wheat GF- TRANS_B102,1-1 vs Conv.bred_L88-18 transgenic lines endosperm tissue (14 and 28 days post anthesis-dpa) and in leaf tissue (8 days post germination ?dpg)	
Comment[Submitted Name]	GF- TRANS_B102,1-1 vs Conv.bred_L88-18 wheat lines	
Experimental Design	genetic_modification_design	transcription profiling by array	
Experimental Design Term Source REF	mo	EFO	
Comment[ArrayExpressReleaseDate]	2005-10-05	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-MEXP-309	
Comment[MAGETAB TimeStamp_Version]	2011-06-30 21:10:07 Last Changed Rev: 14857	
Experimental Factor Name	strain_or_line	organism_part	age	
Experimental Factor Type	strain_or_line	organism_part	age	
Experimental Factor Term Source REF	
Person Last Name	Baudo	Powers	Mc Cluskey	Lyons	Holdsworth	Edwards	Shewry	
Person First Name	Maria	Stephen	Melloney	Rebecca	Michael	Keith	Peter	
Person Mid Initials	M	S	M	R	M	K	P	
Person Email	marcela.baudo@bbsrc.ac.uk	
Person Phone	441582763133	
Person Fax	441582763010	
Person Address	West Common, Harpenden, Hertfordshire, AL52JQ, United Kingdom	
Person Affiliation	Crop Performance and Improvement	
Person Roles	submitter	
Person Roles Term Source REF		
Quality Control Type	spike_quality_control	biological_replicate	dye_swap_quality_control	
Quality Control Term Source REF		The MGED Ontology	The MGED Ontology	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2005-10-05	
PubMed ID	
Publication DOI	
Publication Author List	 Maria Marcela Baudo; Rebecca Lyons; Stephen Powers; Melloney Mc Cluskey; Keith Edwards; Peter Shewry; Michael Holdsworth	
Publication Title	
Publication Status	
Publication Status Term Source REF	
Experiment Description	The high molecular weight (HMW) subunits of wheat glutenin are synthesised only in the starchy endosperm tissue of the developing wheat grain. We compared the expressed genomes of the transgenic wheat line B102,1-1 (Rooke et al. Transgene inheritance, segregation and expression in bread wheat. Euphytica 129, 301-309 (2003)). Both lines were shown to express the HMW-GS Ax1 gene (Halford, N.G. et al. Analysis of HMW glutenin subunits encoded bychromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality. Theor Appl Genet 83, 373-378 (1992).) to the expressed genome of conventionally bred wheat line L88-18 (Lawrence et al. Dough and baking quality of wheat lines in glutenin subunits controlled by Glu-A1, Glu-B1 and Glu-D1 loci. J. Cereal. Sci. 7,109-112 (1988))  which results in the same effects on traits. Transcriptomes comparison analysis was performed in endosperm tissue (14 and 28 days post anthesis-dpa) and in leaf tissue (8 days post germination –dpg), respectively. Each of the transcriptome comparisons was performed using three biological replicates (i.e. per line/tissue /developmental stage selected). Hybridisations were performed in reverse dye labelling.Exceptionally, biological replica 2 was only performed for B102,1-1 (green)/L88-18 (red) labelling and not swap 	
Protocol Name	P-MEXP-8020	P-MEXP-7753	P-MEXP-7755	P-MEXP-7756	P-MEXP-7757	P-MEXP-7758	P-MEXP-7760	P-MEXP-7759	P-MEXP-7761	P-MEXP-7764	
Protocol Type	specified_biomaterial_action	grow	pool	pool	nucleic_acid_extraction	nucleic_acid_extraction	labeling	labeling	hybridization	bioassay_data_transformation	
Protocol Description	Transgenic (expressing HMW-GS 1AX1 transgene) and control (non- expressing HMW-GS 1AX1) and also conventional bred line (i.e. L88-18 expressing HMW-GS 1AX1 for two particular experiments were arranged in a balanced row and column design, providing three biological replicates per treatment (wheat line and developmental stage) for microarray hybridizations.Plants were visually monitored and tagged at the onset of anthesis, and grain samples were collected at 14 and 28 dpa. For leaf samples, plants were harvested at 8 dpg. Seed endosperm (target tissue) was manually dissected out of the embryo and maternal tissue in aseptic conditions. Both, endosperm and leaf samples, when collected, where immediately frozen in liquid nitrogen and stored in -80°C freezer.  	Hexaploid wheat plants were grown in containment glasshouse compartments under controlled environmental conditions (18-20°C/10-14°C day/night; 16 hours-day/8 hours-night, 50-70% humidity, 750 µE/s/m2 irradiance) and with automatic watering. Transgenic and control, non-transformed wheat lines selected for the different transcriptome comparisons studies were grown in parallel under identical environmental conditions. Plants were grown in pots and arranged in a balanced row and column design, providing three biological replicates per treatment (wheat line and developmental stage) for microarray hybridizations. Two plants were grown per pot because two grain developmental stages were studied (14 and 28 days post-anthesis -dpa). Two tillings were kept per plant. A third plant was grown in selected pots for leaf tissue sampling at 8 days post germination (dpg). Plants were visually monitored and tagged at the onset of anthesis, and grain samples were collected at 14 and 28 dpa. For leaf samples, plants were harvested at 8 dpg. Seed endosperm (target tissue) was manually dissected out of the embryo and maternal tissue. Both grain endosperm and expanded leaf tissues were used for total RNA extraction. 	Total RNA from endosperm (3 biological replicates/40 tillings per BR/10-12 grains per ear) was extracted using the method described by Chang et al. (1993) (Chang, S., Puryear, J. & Cairney, J. A Simple and Efficient Method for Isolating RNA from Pine Trees. Plant Molecular Biology Reporter 11, 113-116). Total RNAs were DNase treated (DNase Free kit, Ambion). Integrity of the RNA was checked in formaldehyde agarose gels and quantified using Agilent 2100 Bioanalyzer (RNA 6000 Nano Lab Chip Kit, www.agilent.com). cDNA probes used to hybridize microarray slides were generated from total RNA reversed transcribed and secondary conjugated to amino-allyl fluor dyes Alexa 555 and 647 (Molecular Probes Inc., USA).  Poly(A)+ cDNA transcripts corresponding to mammal-specific mRNA sequences were used as spiking controls and obtained as recombinant DNAs in the plasmid vector pT7T3D-Pac from the maize Gene Discovery Project (http//:www.zmdb.iastate.edu/). The list includes clones pHUM2 (GB: AA418251), pHUM4 (GB:AA464627) and TRAF1 (GB:BC024145). After spiking with different control mRNA transcripts at various concentrations, total RNA samples were reversed transcribed using an oligo(dT)23 anchor primer (Sigma-Genosis) and Superscript III reverse transcriptase (Invitrogen) in the presence of 5-(3-aminoallyl)-2’-deoxiuridine 5’ triphosphate (AA-dUTP) (Sigma) in order to produce aminoallyl-labelled first strand cDNAs (aa-dUTP-cDNA). Steps 1- Priming reaction a. 100 µg total RNA (included spinking control RNA), 8 µl Oligo (dT) 23 anchored primer–0.5 µg/µl/70uM and H2O till 28 ul final volumen. b. Incubation : 70°C/10 min (denaturation). Place on ice 5 min.  2- Fisrt strand cDNA synthesis a- 10µl (5 x First strand Buffer), 5µl (1 M DTT), 2µl (50x aa-dNTP mix*),2µl (SuperScript III Reverse Transcriptase 200U/µl) and H2O. b- Add in 22 µl of priming mix. c- Incubate at 42°C for 2-3 hours. 	Total RNA from leaves (3 biological replicates) was extracted following the method described by Cheng et al.(2001) (Cheng, G.P., Wilson, I.D., Kim, S.H. & Grierson, D. Inhibiting expression of a tomato ripening-associated membrane protein increases organic acids and reduces sugar levels of fruit. Planta 212, 799-807 (2001)), except that the extraction buffer used by these authors was replaced by SDS/phenol buffer. Also, much of the  cell material complexed with SDS was removed from the homogenate before phenol/CHCl3 extraction by the addition of 0.2 volumes of 5M KAc and centrifugation. Total RNAs were DNase treated (DNase kit, Ambion). Integrity of the RNA was checked in formaldehyde agarose gels and quantified using Agilent 2100 Bioanalyzer (RNA 6000 Nano Lab Chip Kit, www.agilent.com). cDNA probes used to hybridize microarray slides were generated from total RNA reversed transcribed and secondary conjugated to amino-allyl fluor dyes Alexa 555 and 647 (Molecular Probes Inc., USA). Poly(A)+ cDNA transcripts corresponding to mammal-specific mRNA sequences were used as spiking controls and obtained as recombinant DNAs in the plasmid vector pT7T3D-Pac from the maize Gene Discovery Project (http//:www.zmdb.iastate.edu/). The list includes clones pHUM2 (GB: AA418251), pHUM4 (GB:AA464627) and TRAF1 (GB:BC024145. After spiking with different control mRNA transcripts at various concentrations, total RNA samples were reversed transcribed using an oligo(dT)23 anchor primer (Sigma-Genosis) and Superscript III reverse transcriptase (Invitrogen) in the presence of 5-(3-aminoallyl)-2’-deoxiuridine 5’ triphosphate (AA-dUTP) (Sigma) in order to produce aminoallyl-labelled first strand cDNAs (aa-dUTP-cDNA). Steps 1- Priming reaction a. 100 µg total RNA, 8 µl Oligo (dT) 23 anchored primer–0.5 µg/µl/70uM and H2O till 28 ul final volumen. b. Incubation : 70°C/10 min (denaturation). Place on ice 5 min. 2- Fisrt strand cDNA synthesis a- 10µl (5 x First strand Buffer), 5µl (1 M DTT), 2µl (50x aa-dNTP mix*),2µl (SuperScript III Reverse Transcriptase 200U/µl) and H2O. b- Add in 22 µl of priming mix. c- Incubate at 42°C for 2-3 hours.  	Total RNA from endosperm (3 biological replicates) was extracted using the method described by Chang et al. (1993) (Chang, S., Puryear, J. & Cairney, J. A Simple and Efficient Method for Isolating RNA from Pine Trees. Plant Molecular Biology Reporter 11, 113-116). Total RNAs were DNase treated (DNase kit, Ambion). Integrity of the RNA was checked in formaldehyde agarose gels and quantified using Agilent 2100 Bioanalyzer (RNA 6000 Nano Lab Chip Kit, www.agilent.com). cDNA probes used to hybridize microarray slides were generated from total RNA reversed transcribed and secondary conjugated to amino-allyl fluor dyes Alexa 555 and 647 (Molecular Probes Inc., USA). Poly(A)+ cDNA transcripts corresponding to mammal-specific mRNA sequences were used as spiking controls . The list includes clones pHUM2 (GB: AA418251), pHUM4 (GB:AA464627) and TRAF1 (GB:BC024145).After spiking with different control mRNA transcripts at various concentrations, total RNA samples were reversed transcribed using an oligo(dT)23 anchor primer (Sigma-Genosis) and Superscript III reverse transcriptase (Invitrogen) in the presence of 5-(3-aminoallyl)-2’-deoxiuridine 5’ triphosphate (AA-dUTP) (Sigma) in order to produce aminoallyl-labelled first strand cDNAs (aa-dUTP-cDNA). Steps 1- Priming reaction a. 100 µg total RNA, 8 µl Oligo (dT) 23 anchored primer–0.5 µg/µl/70uM and H2O till 28 ul final volumen. b. Incubation : 70°C/10 min (denaturation). Place on ice 5 min. 2- Fisrt strand cDNA synthesis a- 10µl (5 x First strand Buffer), 5µl (1 M DTT), 2µl (50x aa-dNTP mix),2µl (SuperScript III Reverse Transcriptase 200U/µl) and H2O. b- Add in 22 µl of priming mix. c- Incubate at 42°C for 2-3 hours. 	Total RNA from leaves was extracted following the method described by Cheng et al.(2001) (Cheng, G.P., Wilson, I.D., Kim, S.H. & Grierson, D. Inhibiting expression of a tomato ripening-associated membrane protein increases organic acids and reduces sugar levels of fruit. Planta 212, 799-807 (2001)), except that the extraction buffer used by these authors was replaced by SDS/phenol buffer. Also, much of the  cell material complexed with SDS was removed from the homogenate before phenol/CHCl3 extraction by the addition of 0.2 volumes of 5M KAc and centrifugation. Total RNAs were DNase treated (DNase kit, Ambion). Integrity of the RNA was checked in formaldehyde agarose gels and quantified using Agilent 2100 Bioanalyzer (RNA 6000 Nano Lab Chip Kit, www.agilent.com). Poly(A)+ cDNA transcripts corresponding to mammal-specific mRNA sequences were used as spiking . The list includes clones pHUM2 (GB: AA418251), pHUM4 (GB:AA464627 and TRAF1 (GB:BC024145). After spiking with different control mRNA transcripts at various concentrations, total RNA samples were reversed transcribed using an oligo(dT)23 anchor primer (Sigma-Genosis) and Superscript III reverse transcriptase (Invitrogen) in the presence of 5-(3-aminoallyl)-2’-deoxiuridine 5’ triphosphate (AA-dUTP) (Sigma) in order to produce aminoallyl-labelled first strand cDNAs (aa-dUTP-cDNA). Steps 1- Priming reaction a. 100 µg total RNA, 8 µl Oligo (dT) 23 anchored primer–0.5 µg/µl/70uM and H2O till 28 ul final volumen. b. Incubation : 70°C/10 min (denaturation). Place on ice 5 min. 2- Fisrt strand cDNA synthesis a- 10µl (5 x First strand Buffer), 5µl (1 M DTT), 2µl (50x aa-dNTP mix),2µl (SuperScript III Reverse Transcriptase 200U/µl) and H2O. b- Add in 22 µl of priming mix. c- Incubate at 42°C for 2-3 hours. The aa-dUTP-cDNAs mix was Hydrolyse and Neutralize: 50 µl (cDNA product), 20 µl (0.5M EDTA), 20 µl (1M NaOH) and H2O till 100 µl. The reaction mix was inclubated at 65°C/10 min. The hydrolised reaction was then transferred to a clean tube containing 20 µl 1M HCl. The aa-dUTP-cDNA was purified using MiniElute spin columns (Qiagen). To elute cDNA, 10 ul of H20 was added to the centre of the column membrane and lets stand for 1 min and then centrifugate for 1 min. Elution was repeated with 10 ul EB buffer (MiniElute, Quiagen) to avoid Tris.  The cDNA used for microarray slides were hybridized in reversed dye (Alexa 647 and 555).  	cDNA probes used to hybridize microarray slides were generated from total RNA (from endosperm or leaf), reversed transcribed and secondary conjugated to amino-allyl fluor dyes (Alexa 555 or 647) (Molecular Probes Inc., USA). cDNA microarray slides were hybridized in reversed dye (alexa 647 and 555). The purified cDNA was split into two for reversed dye labelling. cDNA labelling with Alexa 555 fluor dye 1-To 5 µl aa-dUTP-cDNA was added 3µl (1M sodium bicarbonate labelling Buffer-pH 9), 2 µl Alexa Fluor dye 555 (Molecular Probes) dissolved in DMSO. The reaction mix (10 µl) was incubated in dark for 1 hour at RT.  2-The uncoupled dye material was removed from Alexa dye 555 labelled cDNA: a.40 µl H20 was added to the reaction mix (10 µl) b.5 volumes (250 µl) of PB Buffer (Quiagen) was added to the reaction mix c.The sample was applied to MinElute column (Quiagen) to bind cDNA. d.Column was washed with 750 µl (2 washes with 75% ethanol) and then centrifuged for 1 min. e.The MinElute column was placed in a clean 1.5 ml microcentrifuge tube. f.The cDNA-coupled with Alexa dye 555 was eluted with 10 µl applied to the centre of the columns membrane. After 5 min the column was centrifuge for 5 min. The elution step was then repeated.   	cDNA probes used to hybridize microarray slides were generated from total RNA (from endosperm or leaf), reversed transcribed and secondary conjugated to amino-allyl fluor dyes (Alexa 555 or 647) (Molecular Probes Inc., USA).  cDNA microarray slides were hybridized in reversed dye (alexa 647 and 555). The purified cDNA was split into two for reversed dye labelling. cDNA labelling with Alexa 647 dye: 1-To 5 µl aa-dUTP-cDNA was added 3µl (1M sodium bicarbonate labelling Buffer-pH 9), 2 µl Alexa Fluor dye 647 (Molecular Probes) dissolved in DMSO. The reaction mix (10 µl) was incubated in dark for 1 hour at RT.  2-The uncoupled dye material was removed from Alexa dye 647 labelled cDNA: a.40 µl H20 was added to the reaction mix (10 µl) b.5 volumes (250 µl) of PB Buffer (Quiagen) was added to the reaction mix c.The sample was applied to MinElute column (Quiagen) to bind cDNA. d.Column was washed with 750 µl (2 washes with 75% ethanol) and then centrifuged for 1 min. e.The MinElute column was placed in a clean 1.5 ml microcentrifuge tube. f.The cDNA-coupled with Alexa dye 647 was eluted with 10 µl applied to the centre of the columns membrane. After 5 min the column was centrifuge for 5 min. The elution step was then repeated.   	Hybridization steps: 1-aa-dUTP-cDNA labelled with Alexa dye 555 and 647 (100 ug total labelled cDNA with Alexa 647 and 555 contained in 23 ul  final volume ) were mixed with 25 µl of 2 x Hybridisation mix (400µl 50%Formamide+ 4.5 ml 10xSSC+ 16µl 0.2%SDS) and 2 µl of 1 µg/µl poly(dA).  2-The reaction mix incubated at 95°C/ 3 min to denature the probe.  3-The labelled probe was then applied to the coverslip and the slides placed face down in the hybridisation chamber) 4-The hybridisation incubation was performed overnight at 42°C. 5-Washes: a.Each slide (washes for each of two reversed dyed labelled were performed in parallel) was placed in a falcon 50 ml tube containing Wash solution A (2x SSC/1%SDS) and inverted at RT and cover slip removed. b.Then slide was transferred to a second tube containing fresh Wash solution A and inverted for 15 min/RT c.Then slide transferred to a third tube containing Wash solution B (1x SSC/0.2 %SDS) and inverted for 8 min/ RT d.Then slide transferred to a final tube containing Wash solution C (0.1x SSC/0.2 %SDS) and inverted for 5 min/RT. e.Finally the slide was placed in a dry clean falcon tube and spin immediately at 2,000 rpm to dry. 	Data set from each of performed experiment was exported from GenePix to GeneSpring (GeneSpring 6.2, Silicon Genetics, USA) for normalization using a locally weighted scatter plot smooth (LOWESS) method to ensure that no undesirable relationship in the log2 dye ratio (differential expression) versus log2 dye product (intensity) plots ocurred. The normalized data were then analyzed using GenStat (GenStat 7th Edition, GenStat Procedure Library, Release PL15, Lawes Agricultural Trust, Rothamsted, Harpenden, UK) through fitting a linear mixed model by the method of Residual Maximum Likelihood (REML) (Patterson, H.D. & Thompson, R. Recovery of inter-block information when block-size are unequal. Biometrika 58, 545-554 (1971)). This took account of terms relating to the variance structure of the data: experiment (for transgenic wheat line  versus non-transformed wheat line comparison), biological replicate and technical replicate (dye swap); and included a fixed term for genes. Using “experiment” as a fixed rather than a random model term allowed a test to be made as to whether, when pooling the data from the two separate experiments for transgenic line versus control line, the experiment effect was due to an actual treatment or to design variation. Comparison of model deviance (McCullogh, P. & Nelder, J.A. Generalized linear models. (Chapman and Hall, London, UK; 1989)) confirmed the best form for the random part of each model, the change in deviance being asymptotically distributed as chi-squared on the degrees of freedom associated with the model change. The ratio of each gene effect to its standard error, giving a t-statistic on the model residual degrees of freedom, enabled the significance of the differential expression from 0 (on the log2 scale) to be assessed. Genes with significant differential expression (p <0.05) were filtered on expression (>1.5-fold, >2-fold >3-fold, etc.) and then on the number of replicates present (non-missing observations) for each gene.	
Protocol Parameters					Amplification;Extracted product;	Amplification;Extracted product;	Amplification;Label used;Amount of nucleic acid labeled;	Amount of nucleic acid labeled;Label used;Amplification;	Volume;time;temperature;Quantity of label target used;Chamber type;	
Protocol Hardware	
Protocol Software	
Protocol Contact	
Protocol Term Source REF			mo	mo	mo	mo	mo	mo	mo	mo	
SDRF File	E-MEXP-309.sdrf.txt	
Term Source Name	mo		tair_dev:1.27	The MGED Ontology	ArrayExpress	The MGED Ontology	mo	EFO	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.arabidopsis.org/info/ontologies/	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version			1.27