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Experiment: Effects of the cucumber mosaic virus 2b silencing suppressor protein on inducible plant defence pathways

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-415

In this study, we have examined the effects of the cucumber mosaic virus 2b protein on the salicylic acid (SA) and methyl jasmonate (meJa) induced defence pathways in A. thaliana. We have also investigated the effects of the 2b protein on antimycin A-induced defence, which represents a virus-specific branch of SA-induced defence. Transgenic A. thaliana (ecotype Col 0) expressing the 2b gene from CMV (strain Fny) were treated independently with SA, meJa and AA, as were wild-type controls. After 25 hours, RNA was extracted from aerial tissue for comparison of gene expression profiles in the presence and absence of the 2b protein. The 2b protein is known to suppress SA-induced resistance to plant viruses as well as the siRNA and miRNA-mediated gene silencing pathways. It is able to suppress SA-mediated induction of alternative oxidase, a gene that functions in the AA-induced virus-specific branch of SA-induced defence (Ji and Ding, 2001, Mol. Plant Microbe Interactions 14, 715-724). Furthermore, the 2b protein is the major determinant of viral symptoms in CMV and causes symptom-like alterations in the phenotype of Arabidopsis thaliana plants expressing 2b transgenes (Lewsey et al., 2007, Plant J., in press). However, its effects on the meJa-induced defence pathway, which has crosstalk with the SA-induced defence pathway, are not known.

About the Experimenter

Name:Dr Mathew Lewsey
Head of Lab Name:Dr John P Carr
Lab:
Institute: Department of Plant Sciences
Address:Molecular Virology
Department of Plant Sciences
Downing Street
Cambridge
Postcode: CB2 3EA
Country: UK
 
Telephone Number: 01223330215

All of the data available in this website/database is free, and you are free to do whatever you please with it. If you intend to publish work based on any of this data, please acknowledge us, contact the experimenter above, and either acknowledge them or use them as co-authors in the work.

About this Experiment

Experiment Type: compound_based_treatment_design;genetic_modification_design
Number of Slides:24
 
Experimental Parameters:
parametercompound_based_treatment
parameterindividual_genetic_characterisics
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Lewsey: Effects of the cucumber mosaic virus 2b silencing suppressor protein on inducible plant defence pathways_genome

Slide: Lewsey_1-12_Fny-CMV2b-salicylic-acid_Rep2_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-12_Fny-CMV2b-salicylic-acid_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-12_Fny-CMV2b-salicylic-acid_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 1mM salicylic acid and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionSalicylic acid sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.769543647766
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.61
NoiseAvg:1.67,Stdev:0.04,Max:1.8,Min:1.5
Central-Avg:5485,Count:9
Corner+Avg:67,Count:32
Corner-Avg:7417,Count:32
BackgroundAvg:43.67,Stdev:0.51,Max:45.0,Min:42.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.769543647766
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-11_Wild-type-salicylic-acid_Rep2_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-11_Wild-type-salicylic-acid_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-11_Wild-type-salicylic-acid_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 1mM salicylic acid and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction
Additional Organism Information:
Sample descriptionSalicylic acid sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:3.30672
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.80
NoiseAvg:1.82,Stdev:0.06,Max:2.0,Min:1.7
BackgroundAvg:51.37,Stdev:0.51,Max:52.8,Min:50.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF3.306720
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-10_Fny-CMV2b-H2O_Rep2_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-10_Fny-CMV2b-H2O_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-10_Fny-CMV2b-H2O_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionWater sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.091073
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.63
NoiseAvg:1.73,Stdev:0.05,Max:1.9,Min:1.6
BackgroundAvg:46.21,Stdev:0.39,Max:47.1,Min:44.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.091073
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-9_Wild-type-H2O_Rep2_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-9_Wild-type-H2O_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-9_Wild-type-H2O_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionWater sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.855461
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.55
NoiseAvg:1.60,Stdev:0.05,Max:1.8,Min:1.4
BackgroundAvg:43.87,Stdev:0.26,Max:44.4,Min:43.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.855461
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-8_Fny-CMV2b-methyl-jasmonate_Rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-8_Fny-CMV2b-methyl-jasmonate_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-8_Fny-CMV2b-methyl-jasmonate_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 250 micromolar methyl jasmonate and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionMethyl jasmonate sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.927729606628
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.45
NoiseAvg:1.47,Stdev:0.06,Max:1.8,Min:1.3
Central-Avg:5166,Count:9
Corner+Avg:56,Count:32
Corner-Avg:6606,Count:32
BackgroundAvg:41.07,Stdev:0.19,Max:41.5,Min:40.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.927729606628
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-7_Wild-type-methyl-jasmonate_Rep1_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-7_Wild-type-methyl-jasmonate_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-7_Wild-type-methyl-jasmonate_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 250 micromolar methyl jasmonate and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionMethyl jasmonate sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.508786201477
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.96
NoiseAvg:2.32,Stdev:0.50,Max:5.4,Min:2.0
Central-Avg:5178,Count:9
Corner+Avg:55,Count:32
Corner-Avg:6017,Count:32
BackgroundAvg:56.41,Stdev:0.47,Max:58.0,Min:55.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.508786201477
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-6_Fny-CMV2b-antimycin-A_Rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-6_Fny-CMV2b-antimycin-A_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-6_Fny-CMV2b-antimycin-A_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 50 micromolar antimycin A and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionAntimycin A sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants..
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:3.692595720291
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.53
NoiseAvg:1.52,Stdev:0.04,Max:1.7,Min:1.4
Central-Avg:5578,Count:9
Corner+Avg:51,Count:32
Corner-Avg:6298,Count:32
BackgroundAvg:41.81,Stdev:0.22,Max:42.3,Min:41.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF3.692595720291
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-5_Wild-type-antimycinA_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-5_Wild-type-antimycinA_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-5_Wild-type-antimycinA_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 50 micromolar antimycin A and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionAntimycin A sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants..
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.705045
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.92
NoiseAvg:2.13,Stdev:0.11,Max:2.6,Min:1.9
BackgroundAvg:55.80,Stdev:0.53,Max:57.5,Min:54.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.705045
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-4_Fny-CMV2b-salicylic-acid_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-4_Fny-CMV2b-salicylic-acid_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-4_Fny-CMV2b-salicylic-acid_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 1mM salicylic acid and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionSalicylic acid sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.314213037491
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.52
NoiseAvg:1.63,Stdev:0.07,Max:1.9,Min:1.5
Central-Avg:5804,Count:9
Corner+Avg:61,Count:32
Corner-Avg:7249,Count:32
BackgroundAvg:42.30,Stdev:0.31,Max:43.1,Min:41.6
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.314213037491
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-3_Wild-type-salicylic-acid_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-3_Wild-type-salicylic-acid_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-3_Wild-type-salicylic-acid_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 1mM salicylic acid and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction
Additional Organism Information:
Sample descriptionSalicylic acid sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.543917
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.47
NoiseAvg:1.49,Stdev:0.07,Max:1.7,Min:1.3
BackgroundAvg:40.80,Stdev:0.12,Max:41.1,Min:40.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.543917
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-2_Fny-CMV2b-H2O_Rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-2_Fny-CMV2b-H2O_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-2_Fny-CMV2b-H2O_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionWater sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.337679
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.54
NoiseAvg:1.82,Stdev:0.09,Max:2.3,Min:1.6
BackgroundAvg:44.07,Stdev:0.43,Max:45.4,Min:43.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.337679
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-1_Wild-type-H2O_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-1_Wild-type-H2O_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-1_Wild-type-H2O_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionWater sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.994913
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.71
NoiseAvg:1.83,Stdev:0.08,Max:2.1,Min:1.6
BackgroundAvg:47.30,Stdev:0.57,Max:49.4,Min:46.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.994913
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-13_Wild-type-antimycinA_Rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-13_Wild-type-antimycinA_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-13_Wild-type-antimycinA_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 50 micromolar antimycin A and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionAntimycin A sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants..
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.765096
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.86
NoiseAvg:1.95,Stdev:0.03,Max:2.1,Min:1.9
BackgroundAvg:53.45,Stdev:0.36,Max:54.1,Min:52.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.765096
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-14_Fny-CMV2b-antimycin-A_Rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-14_Fny-CMV2b-antimycin-A_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-14_Fny-CMV2b-antimycin-A_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 50 micromolar antimycin A and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionAntimycin A sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants..
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:3.241312265396
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.38
NoiseAvg:1.37,Stdev:0.05,Max:1.5,Min:1.2
Central-Avg:4609,Count:9
Corner+Avg:44,Count:32
Corner-Avg:5495,Count:32
BackgroundAvg:38.59,Stdev:0.34,Max:39.4,Min:37.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF3.241312265396
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-15_Wild-type-methyl-jasmonate_Rep2_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-15_Wild-type-methyl-jasmonate_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-15_Wild-type-methyl-jasmonate_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 250 micromolar methyl jasmonate and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionMethyl jasmonate sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.973613858223
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.75
NoiseAvg:1.88,Stdev:0.07,Max:2.3,Min:1.8
Central-Avg:5277,Count:9
Corner+Avg:58,Count:32
Corner-Avg:6431,Count:32
BackgroundAvg:49.46,Stdev:0.88,Max:52.0,Min:48.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.973613858223
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-16_Fny-CMV2b-methyl-jasmonate_Rep2_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-16_Fny-CMV2b-methyl-jasmonate_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-16_Fny-CMV2b-methyl-jasmonate_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 250 micromolar methyl jasmonate and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionMethyl jasmonate sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.975891828537
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.56
NoiseAvg:1.52,Stdev:0.05,Max:1.7,Min:1.4
Central-Avg:5782,Count:9
Corner+Avg:53,Count:32
Corner-Avg:6838,Count:32
BackgroundAvg:44.85,Stdev:0.35,Max:45.7,Min:43.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.975891828537
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-17_Wild-type-H2O_Rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-17_Wild-type-H2O_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-17_Wild-type-H2O_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionWater sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.919992
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.77
NoiseAvg:1.85,Stdev:0.04,Max:2.0,Min:1.7
BackgroundAvg:49.45,Stdev:0.37,Max:50.3,Min:48.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.919992
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-18_Fny-CMV2b-H2O_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-18_Fny-CMV2b-H2O_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-18_Fny-CMV2b-H2O_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionWater sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.019186
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.89
NoiseAvg:2.02,Stdev:0.07,Max:2.2,Min:1.8
BackgroundAvg:54.93,Stdev:0.45,Max:55.9,Min:53.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.019186
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-19_Wild-type-salicylic-acid_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-19_Wild-type-salicylic-acid_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-19_Wild-type-salicylic-acid_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 1mM salicylic acid and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction
Additional Organism Information:
Sample descriptionSalicylic acid sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.10152
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.39
NoiseAvg:1.54,Stdev:0.06,Max:1.7,Min:1.4
BackgroundAvg:39.98,Stdev:0.30,Max:40.9,Min:39.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.101520
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-20_Fny-CMV2b-salicylic-acid_Rep3_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-20_Fny-CMV2b-salicylic-acid_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-20_Fny-CMV2b-salicylic-acid_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 1mM salicylic acid and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionSalicylic acid sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.69446182251
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.44
NoiseAvg:1.45,Stdev:0.05,Max:1.7,Min:1.3
Central-Avg:5061,Count:9
Corner+Avg:50,Count:32
Corner-Avg:6030,Count:32
BackgroundAvg:39.82,Stdev:0.24,Max:40.5,Min:39.4
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.694461822510
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-21_Wild-type-antimycinA_Rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-21_Wild-type-antimycinA_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-21_Wild-type-antimycinA_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 50 micromolar antimycin A and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionAntimycin A sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants..
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.46278
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.62
NoiseAvg:1.70,Stdev:0.05,Max:1.8,Min:1.5
BackgroundAvg:45.14,Stdev:0.39,Max:46.0,Min:44.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.462780
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-22_Fny-CMV2b-antimycin-A_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-22_Fny-CMV2b-antimycin-A_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-22_Fny-CMV2b-antimycin-A_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 50 micromolar antimycin A and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionAntimycin A sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants..
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:3.479066133499
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.47
NoiseAvg:1.42,Stdev:0.04,Max:1.5,Min:1.3
Central-Avg:4935,Count:9
Corner+Avg:49,Count:32
Corner-Avg:5928,Count:32
BackgroundAvg:41.03,Stdev:0.37,Max:42.1,Min:40.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF3.479066133499
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-23_Wild-type-methyl-jasmonate_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-23_Wild-type-methyl-jasmonate_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-23_Wild-type-methyl-jasmonate_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Columbia 0
Stock Code:
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 250 micromolar methyl jasmonate and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionMethyl jasmonate sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.067264080048
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.85
NoiseAvg:1.94,Stdev:0.05,Max:2.1,Min:1.8
Central-Avg:5884,Count:9
Corner+Avg:61,Count:32
Corner-Avg:7103,Count:32
BackgroundAvg:53.11,Stdev:0.46,Max:54.3,Min:52.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.067264080048
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lewsey_1-24_Fny-CMV2b-methyl-jasmonate_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Lewsey_1-24_Fny-CMV2b-methyl-jasmonate_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lewsey_1-24_Fny-CMV2b-methyl-jasmonate_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Fny 2b 2.11C
Stock Code:
Genetic Background: Col-0
Growth Conditions:
Average temperature21oC
Average humidity60%
LocationGrowth Room
Growth substrate(Source: Levington M3 compost (Scotts, Chilworth, Ipswich, UK))Natural soil
StratificationFive days in dark at 4 degrees celsius.
Additional informationSeeds were germinated on half MS medium plates containing 1% (w/v) sucrose and 1% (w/v) agar, then transplanted onto soil seven days after germination.
SterilisationTwo minutes in 70% (v/v) ethanol followed by ten minutes in a solution of 0.4% (w/v) sodium hypochlorite and 0.5% (w/v) SDS
Growth protocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
WateringWatered daily until soil was moist
Developmental Stage:
Developmental stage(Source: Paradigm Genetics: Boyes Key)3.50
Genetic Variation: TDNA gene knock out
Tissue: all aerial tissues
in vivo Treatment:
Treatment protocolApproximately ten plants were sprayed with water containing 250 micromolar methyl jasmonate and 0.1% (v/v) ethanol, 5.5 hrs into the light cycle, when plants were 6 weeks old (growth stage 3.50 to 3.70). Plant tissues were harvested 25 hours after treatment and pooled to minimise variation. The pooled tissue was then used for RNA extraction.
Additional Organism Information:
Sample descriptionMethyl jasmonate sprayed A.thaliana plants. Aerial tissue pooled from approximately ten plants.
Other Information:
Allele35S expression of Cucumber mosaic virus 2b gene

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.700993180275
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.77
NoiseAvg:1.89,Stdev:0.07,Max:2.1,Min:1.7
Central-Avg:4786,Count:9
Corner+Avg:60,Count:32
Corner-Avg:6787,Count:32
BackgroundAvg:49.00,Stdev:0.72,Max:50.5,Min:47.4
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.700993180275
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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