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Experiment: Transcriptome changes of Arabidopsis during pathogen and insect attack

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-330

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, P. rapae, and F. occidentalis, more than 50% were also induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker-specific. All together our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

In the experimental set up we intended to select for genes that showed a more than 2-fold change in the same direction(up or down)at two time points after pathogen or insect attack.

The results are described in the following paper:

De Vos, M., Van Oosten, V.R., Van Poecke, R.M.P., Van Pelt, J.A., Pozo, M.J., Mueller, M.J., Buchala, A.J., Metraux, J.-P., Van Loon, L.C., Dicke, M. and Pieterse, C.M.J. (2005). Signal signature and transcriptome changes in Arabidopsis upon pathogen and insect attack. Molecular Plant-Microbe Interactions, in press.

About the Experimenter

Name:Prof. Corne Pieterse
Head of Lab Name:Prof Corne Pieterse
Lab: Section Phytopathology
Institute: Utrecht University
Address:Department of Biology
Utrecht University
Sorbonnelaan 16
Utrecht
Postcode: 3584CA
Country: The Netherlands
 
Telephone Number: +31 30 253 3013
Fax Number: +31 30 251 8366

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: time_series_design; pathogenicity_design
Number of Slides:14
 
Experimental Parameters:
parameter infect
parameter timepoint
Quality Control Measures Taken:
References:
referenceDe Vos, M., Van Oosten, V.R., Van Poecke, R.M.P., Van Pelt, J.A., Pozo, M.J., Mueller, M.J., Buchala, A.J., Métraux, J.-P., Van Loon, L.C., Dicke, M. and Pieterse, C.M.J. (2005). Signal signature and transcriptome changes in Arabidopsis upon pathogen and insect attack. Molecular Plant-Microbe Interactions, in press.
 
Other Information:
GEO accessionGSE5525

Slides in this Experiment

Hybridisation Set: Pieterse_set

Slide: Pieterse_1-1_Control-12h_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 ("Pieterse_1-1_Control-12h_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: Pieterse_1-1_Control-12h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 12 hour time point.
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
Separation Technique: Rosette was cut with a razor blade from the root system.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.495009
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:
RawQ2.23
NoiseAvg:3.09,Stdev:0.11,Max:3.5,Min:2.9
BackgroundAvg:51.66,Stdev:1.04,Max:53.5,Min:48.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.495009
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-2_Control-24h_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 ("Pieterse_1-2_Control-24h_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: Pieterse_1-2_Control-24h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 24 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.545493
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:
RawQ1.91
NoiseAvg:2.59,Stdev:0.12,Max:3.0,Min:2.4
BackgroundAvg:42.28,Stdev:0.44,Max:44.0,Min:41.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.545493
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-3_Control-48h_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 ("Pieterse_1-3_Control-48h_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: Pieterse_1-3_Control-48h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 48 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.491924
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:2.74,Stdev:0.10,Max:3.0,Min:2.5
BackgroundAvg:45.53,Stdev:0.43,Max:46.4,Min:43.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.491924
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-4_Control-72h_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 ("Pieterse_1-4_Control-72h_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: Pieterse_1-4_Control-72h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 72 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.524785
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:
RawQ2.01
NoiseAvg:2.63,Stdev:0.06,Max:2.8,Min:2.5
BackgroundAvg:43.85,Stdev:0.47,Max:45.2,Min:42.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.524785
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-5_avrPstDC3000-12h_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 ("Pieterse_1-5_avrPstDC3000-12h_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: Pieterse_1-5_avrPstDC3000-12h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 12 hour time point
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70% relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentP. syringae pv. tomato DC3000 with the plasmid pV288 carrying avirulence gene avrRpt2 (Kunkel et al., 1993) was cultured overnight at 28°C in liquid King's medium B, supplemented with 25 mg.L-1 kanamycin to select for the plasmid. Subsequently, bacterial cells were collected by centrifugation and resuspended in 10 mM MgSO4 to a final density of 107 cfu.mL-1. Wild-type Col-0 plants were inoculated by pressure infiltrating a suspension of P. syringae pv. tomato DC3000(avrRpt2) at 107 cfu.mL-1 into all fully expanded leaves of 5-week-old plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.395876
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:
RawQ2.06
NoiseAvg:2.82,Stdev:0.14,Max:3.2,Min:2.5
BackgroundAvg:43.65,Stdev:0.65,Max:45.8,Min:42.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.395876
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-6_avrPstDC3000-24h_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 ("Pieterse_1-6_avrPstDC3000-24h_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: Pieterse_1-6_avrPstDC3000-24h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 24 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentP. syringae pv. tomato DC3000 with the plasmid pV288 carrying avirulence gene avrRpt2 (Kunkel et al., 1993) was cultured overnight at 28°C in liquid King's medium B, supplemented with 25 mg.L-1 kanamycin to select for the plasmid. Subsequently, bacterial cells were collected by centrifugation and resuspended in 10 mM MgSO4 to a final density of 107 cfu.mL-1. Wild-type Col-0 plants were inoculated by pressure infiltrating a suspension of P. syringae pv. tomato DC3000(avrRpt2) at 107 cfu.mL-1 into all fully expanded leaves of 5-week-old plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.675286
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:
RawQ1.81
NoiseAvg:2.20,Stdev:0.12,Max:2.5,Min:2.0
BackgroundAvg:39.25,Stdev:0.59,Max:40.4,Min:37.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.675286
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-7_Abrassicicola-24h_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 ("Pieterse_1-7_Abrassicicola-24h_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: Pieterse_1-7_Abrassicicola-24h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pad3-1
Stock Code: N3805
Age: 24 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentBioassays with the fungal leaf pathogen Alternaria brassicicola strain MUCL 20297 were carried out as described by Ton et al. (MPMI, 2002). Briefly, A. brassicicola was grown on potato dextrose agar plates for 2 weeks at 22°C. Subsequently, conidia were collected as described by Broekaert et al. (1990). Five-week-old susceptible pad3-1 plants were challenge inoculated by applying 3-µL drops of 10 mM MgSO4 containing 106 spores per mL onto all fully expanded leaves of 5-week-old plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.769256
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:
RawQ1.72
NoiseAvg:2.08,Stdev:0.08,Max:2.5,Min:1.9
BackgroundAvg:39.02,Stdev:0.33,Max:39.9,Min:38.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.769256
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-8_Abrassicicola-48h_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 ("Pieterse_1-8_Abrassicicola-48h_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: Pieterse_1-8_Abrassicicola-48h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pad3-1
Stock Code: N3805
Age: 48 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentBioassays with the fungal leaf pathogen Alternaria brassicicola strain MUCL 20297 were carried out as described by Ton et al. (MPMI, 2002). Briefly, A. brassicicola was grown on potato dextrose agar plates for 2 weeks at 22°C. Subsequently, conidia were collected as described by Broekaert et al. (1990). Five-week-old susceptible pad3-1 plants were challenge inoculated by applying 3-µL drops of 10 mM MgSO4 containing 106 spores per mL onto all fully expanded leaves of 5-week-old plants.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.725261
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:
RawQ1.74
NoiseAvg:2.08,Stdev:0.06,Max:2.3,Min:1.9
BackgroundAvg:41.58,Stdev:0.30,Max:42.2,Min:40.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.725261
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-9_Prapae-12h_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 ("Pieterse_1-9_Prapae-12h_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: Pieterse_1-9_Prapae-12h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 12 hour time point
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentTissue-chewing larvae of the small cabbage white butterfly Pieris rapae were reared on Brussels sprout plants (Brassica oleracea gemmifera cv. Cyrus) in a growth chamber with a 16-h day and 8-h night cycle (21°C; 50-70% relative humidity), as described previously (Van Poecke et al., 2001). Infestation of Arabidopsis Col-0 plants was carried out by transferring five first-instar larvae of P. rapae to each plant using a fine paintbrush.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.889405
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:
RawQ1.69
NoiseAvg:2.32,Stdev:0.16,Max:3.1,Min:2.1
BackgroundAvg:42.19,Stdev:0.62,Max:44.1,Min:40.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.889405
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-10_Prapae-24h_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 ("Pieterse_1-10_Prapae-24h_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: Pieterse_1-10_Prapae-24h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 24 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentTissue-chewing larvae of the small cabbage white butterfly Pieris rapae were reared on Brussels sprout plants (Brassica oleracea gemmifera cv. Cyrus) in a growth chamber with a 16-h day and 8-h night cycle (21°C; 50-70% relative humidity), as described previously (Van Poecke et al., 2001). Infestation of Arabidopsis Col-0 plants was carried out by transferring five first-instar larvae of P. rapae to each plant using a fine paintbrush.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.918456
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:
RawQ1.83
NoiseAvg:2.47,Stdev:0.06,Max:2.7,Min:2.3
BackgroundAvg:44.34,Stdev:0.40,Max:45.5,Min:43.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.918456
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-11_Foccidentalis-24h_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 ("Pieterse_1-11_Foccidentalis-24h_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: Pieterse_1-11_Foccidentalis-24h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 24 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentThe population of the Western flower thrips Frankliniella occidentalis originated from a greenhouse infestation on chrysanthemum. This virus-free population was reared on Phaseolus vulgaris cv. Prelude pods, supplied with Pinus pollen, in glass jars that were placed at 25°C in a growth chamber with a 16-h day and 8-h night cycle as described (Kindt et al., 2003). Thrips infestations were performed by transferring 20 larvae of F. occidentalis to each Arabidopsis Col-0 plant.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.467687
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:
RawQ2.19
NoiseAvg:3.37,Stdev:0.13,Max:4.1,Min:3.1
BackgroundAvg:50.09,Stdev:0.45,Max:51.5,Min:48.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.467687
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-12_Foccidentalis-48h_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 ("Pieterse_1-12_Foccidentalis-48h_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: Pieterse_1-12_Foccidentalis-48h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 48 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentThe population of the Western flower thrips Frankliniella occidentalis originated from a greenhouse infestation on chrysanthemum. This virus-free population was reared on Phaseolus vulgaris cv. Prelude pods, supplied with Pinus pollen, in glass jars that were placed at 25°C in a growth chamber with a 16-h day and 8-h night cycle as described (Kindt et al., 2003). Thrips infestations were performed by transferring 20 larvae of F. occidentalis to each Arabidopsis Col-0 plant.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.761972
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:2.17,Stdev:0.07,Max:2.3,Min:1.9
BackgroundAvg:38.93,Stdev:0.41,Max:40.4,Min:38.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.761972
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-13_Mpersicae-48h_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 ("Pieterse_1-13_Mpersicae-48h_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: Pieterse_1-13_Mpersicae-48h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 48 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentPhloem-feeding green peach aphids (Myzus persicae) were maintained on Brassica chinensis L. cv. Granaat under greenhouse conditions (25°C; 50-70% relative humidity). The 16-h light period prevented sexual reproduction, keeping the population clonal. Arabidopsis Col-0 plants were infested with M. persicae by transferring 40 nymphs and apterous adults to each plant (Van Poecke et al., 2003).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.960164
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:
RawQ1.74
NoiseAvg:2.13,Stdev:0.08,Max:2.4,Min:2.0
BackgroundAvg:40.86,Stdev:0.41,Max:42.0,Min:40.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.960164
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_1-14_Mpersicae-72h_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 ("Pieterse_1-14_Mpersicae-72h_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: Pieterse_1-14_Mpersicae-72h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Age: 72 hour timepoint
Growth Conditions:
Growth protocolSeeds were sown in quartz sand for two weeks. Two-week-old seedlings were transferred to 60-mL pots containing a sand - potting soil mixture that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with a 8-h day (200 uEm-2.sec-1 at 24oC) and 16-h night (20oC) cycle at 70percent relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Diseased: Normal
in vivo Treatment:
TreatmentPhloem-feeding green peach aphids (Myzus persicae) were maintained on Brassica chinensis L. cv. Granaat under greenhouse conditions (25°C; 50-70% relative humidity). The 16-h light period prevented sexual reproduction, keeping the population clonal. Arabidopsis Col-0 plants were infested with M. persicae by transferring 40 nymphs and apterous adults to each plant (Van Poecke et al., 2003).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:1.120775
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:
RawQ2.16
NoiseAvg:2.95,Stdev:0.09,Max:3.1,Min:2.7
BackgroundAvg:49.64,Stdev:0.53,Max:51.0,Min:48.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.120775
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team