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Experiment: Arabidopsis thaliana/Phytophthora parasitica compatible interaction transcriptome

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-468

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to particular physiological characteristics, no treatments against diseases caused by oomycetes are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. The present project is focused on the molecular mechanisms that underlie the compatible plant-oomycete interaction and plant disease.The laboratory developed a novel interaction system involving the model plant, Arabidopsis thaliana and Phytophthora parasitica, a soil-borne pathogen infecting a wide host range, thus representing the majority of Phytophthora species. A characteristic feature of the compatible Arabidopsis/P. parasitica interaction is an extended biotrophic phase, before infection becomes necrotrophic. Because the initial biotrophic phase is extremely short on natural (e.g. solanaceous) hosts, the Arabidopsis system provides the opportunity to analyze, for both interaction partners, the molecular events that determine the initiation of infection and the switch to necrotrophy.The present project aims at analyzing the compatible interaction between A. thaliana roots and P. parasitica. The Affymetrix A. thaliana full genome chip will be used to characterize modulations of the transcriptome occurring over a period of 24h from the onset of plant root infection to the beginning of necrotrophy. Parallel to this study, a custom designed P. parasitica biochip will enable analyzing of P. parasitica gene expression during the same stages.

About the Experimenter

Name: Agnes Attard
Head of Lab Name: Harald Keller
Lab:
Address:400, route des Chappes
BP 167
Postcode: F-06903 Sophia-antipolis
Country: France
 
Telephone Number: 33492386594
Fax Number: 33492386587

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: pathogenicity_design
Number of Slides:10
 
Experimental Parameters:
parameterinfect
Quality Control Measures Taken:
no-plants-pooled
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Attard: Arabidopsis thaliana/Phytophthora parasitica compatible interaction transcriptome_genome

Slide: Attard_1-1_At-Control_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 ("Attard_1-1_At-Control_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: Attard_1-1_At-Control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample Descriptionmock inoculated roots
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.941741406918
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:
RawQ2.42
NoiseAvg:2.51,Stdev:0.08,Max:2.8,Min:2.4
Central-Avg:6106,Count:9
Corner+Avg:73,Count:32
Corner-Avg:7992,Count:32
BackgroundAvg:57.94,Stdev:0.69,Max:59.6,Min:56.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.941741406918
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-2_At-Control_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 ("Attard_1-2_At-Control_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: Attard_1-2_At-Control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample Descriptionmock inoculated roots
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.854700684547
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:
RawQ2.33
NoiseAvg:2.40,Stdev:0.05,Max:2.5,Min:2.3
Central-Avg:6295,Count:9
Corner+Avg:72,Count:32
Corner-Avg:7201,Count:32
BackgroundAvg:56.85,Stdev:0.46,Max:58.0,Min:55.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.854700684547
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-3_At-Pp-2.5-hai_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 ("Attard_1-3_At-Pp-2.5-hai_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: Attard_1-3_At-Pp-2.5-hai_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.684968292713
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:
RawQ2.81
NoiseAvg:3.45,Stdev:0.39,Max:5.8,Min:3.1
Central-Avg:6637,Count:9
Corner+Avg:77,Count:32
Corner-Avg:7494,Count:32
BackgroundAvg:73.50,Stdev:0.66,Max:75.0,Min:71.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.684968292713
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-4_At-Pp-2.5-hai_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 ("Attard_1-4_At-Pp-2.5-hai_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: Attard_1-4_At-Pp-2.5-hai_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.80202049017
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:
RawQ3.45
NoiseAvg:4.01,Stdev:0.08,Max:4.2,Min:3.8
Central-Avg:7116,Count:9
Corner+Avg:92,Count:32
Corner-Avg:7525,Count:32
BackgroundAvg:87.98,Stdev:1.21,Max:90.9,Min:84.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.802020490170
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-5_At-Pp-6-hai_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 ("Attard_1-5_At-Pp-6-hai_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: Attard_1-5_At-Pp-6-hai_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.742000758648
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:
RawQ2.51
NoiseAvg:2.77,Stdev:0.06,Max:2.9,Min:2.6
Central-Avg:7724,Count:9
Corner+Avg:80,Count:32
Corner-Avg:8752,Count:32
BackgroundAvg:62.90,Stdev:0.34,Max:63.7,Min:61.6
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.742000758648
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-6_At-Pp-6-hai_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 ("Attard_1-6_At-Pp-6-hai_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: Attard_1-6_At-Pp-6-hai_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.787004172802
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:
RawQ2.96
NoiseAvg:3.40,Stdev:0.06,Max:3.6,Min:3.2
Central-Avg:6692,Count:9
Corner+Avg:74,Count:32
Corner-Avg:7575,Count:32
BackgroundAvg:77.19,Stdev:0.72,Max:79.7,Min:74.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.787004172802
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-7_At-Pp-10.5-hai_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 ("Attard_1-7_At-Pp-10.5-hai_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: Attard_1-7_At-Pp-10.5-hai_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:0.667812287807
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:
RawQ2.54
NoiseAvg:2.77,Stdev:0.10,Max:3.1,Min:2.5
Central-Avg:8149,Count:9
Corner+Avg:80,Count:32
Corner-Avg:9078,Count:32
BackgroundAvg:62.07,Stdev:0.36,Max:63.1,Min:61.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.667812287807
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-8_At-Pp-10.5-hai_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 ("Attard_1-8_At-Pp-10.5-hai_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: Attard_1-8_At-Pp-10.5-hai_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:1.283706665039
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:
RawQ2.47
NoiseAvg:2.65,Stdev:0.08,Max:2.9,Min:2.5
Central-Avg:7221,Count:9
Corner+Avg:74,Count:32
Corner-Avg:8149,Count:32
BackgroundAvg:61.39,Stdev:0.40,Max:62.4,Min:59.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.283706665039
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-9_At-Pp-30-hai_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 ("Attard_1-9_At-Pp-30-hai_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: Attard_1-9_At-Pp-30-hai_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:1.773631930351
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:
RawQ2.04
NoiseAvg:1.82,Stdev:0.06,Max:2.0,Min:1.7
Central-Avg:6213,Count:9
Corner+Avg:65,Count:32
Corner-Avg:7612,Count:32
BackgroundAvg:48.12,Stdev:0.68,Max:49.9,Min:46.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.773631930351
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Attard_1-10_At-Pp-30-hai_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 ("Attard_1-10_At-Pp-30-hai_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: Attard_1-10_At-Pp-30-hai_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: CS60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationStratification was performed 2 days at 4°C
SterilisationSeeds were surface sterilised 5 min with sodium hypochlorite (20% commercial bleach; NaOCl, 2.5% active ingredient) and rinsed twice in a 95% ethanol solution.
ProtocolSeeds were grown on 1 x Murashige and skoog agar medium (MS; 4.4 gL-1, Sigma Chemical Compagny, MO, USA) ) supplemented with 10 gL-1 saccharose (Prolabo) and 20 gL-1 agar (difco Agar, Dedon, Dickinson and compagnie, MD, USA). 15 days old plants were transferred to new square Petri dishes containing a strip of 1 x MS agar Medium and underneath 10 ml of 0.1 x MS medium. Plants were grown under 8h photoperiod at 25°C for 1 month.
Percentage Agrose20
Substrate Sterilising Procedure110°c for 20 min
Plant Spacing10 plants per Petri dish
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting(Source: Cool white fluorescent manufactured by Phillips. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: roots
Additional Organism Information:
Sample DescriptionA. thaliana roots were inoculated with Phytophthora parasitica 310 strain (10E6 zoospores per Petri dish
Other Information:
Timecourse start procedureStart time was time when plants were inoculated

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:1.37283539772
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:
RawQ2.13
NoiseAvg:2.07,Stdev:0.07,Max:2.3,Min:1.9
Central-Avg:6367,Count:9
Corner+Avg:72,Count:32
Corner-Avg:8087,Count:32
BackgroundAvg:51.76,Stdev:0.81,Max:53.6,Min:49.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.372835397720
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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