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Experiment: Priming for enhanced MeJA-responsive gene expression in Arabidopsis plants expressing P. fluorescens WCS417r-mediated ISR

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-463

Upon appropriate stimulation, plants can develop an enhanced capacity to express infection-induced cellular defense responses, a phenomenon known as the primed state. Colonization of the roots of Arabidopsis thaliana by the beneficial rhizobacterial strain Pseudomonas fluorescens WCS417r primes the leaf tissue for enhanced pathogen- and insect-induced expression of jasmonate (JA)-responsive genes, resulting in an induced systemic resistance (ISR) that is effective against different types of pathogens and insect herbivores. Here we investigated the molecular mechanism of this rhizobacteria-induced priming response by following a whole-genome transcript profiling approach. To this end we profiled the transcriptome of the leaves of 5-week-old Arabidopsis Col-0 plants at 0, 1, 3, and 6 hours after exogenous application of 50 µM MeJA. This was done with Col-0 plants that were grown in soil with or without ISR-inducing Pseudmonas fluorescens WCS417r rhizobacteria. This microarray analysis is described in Pozo, M.J., Van der Ent, S., Van Loon, L.C. and Pieterse, C.M.J. (2008). The transcription factor MYC2 is involved in priming for enhanced defense during rhizobacteria-induced systemic resistance in Arabidopsis. New Phytologist, in press.

About the Experimenter

Name:Prof. Corne Pieterse
Head of Lab Name: Corne Pieterse
Lab:
Address:Plant-Microbe Interactions
Department of Biology
Utrecht University
Padualaan 8
Utrecht
Postcode: 3584 CH
Country: Netherlands
 
Telephone Number: 0302536887
Fax Number: 0302532837

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

About this Experiment

Experiment Type: compound_treatment_design; time_series_design;pathogenicity_design
Number of Slides:8
 
Experimental Parameters:
parametercompound_based_treatment
parameterinfect
parametertimepoint
Quality Control Measures Taken:
References:
referencePozo, M.J., Van der Ent, S., Van Loon, L.C. and Pieterse, C.M.J. (2008)
 
Other Information:

Slides in this Experiment

Hybridisation Set: Pieterse: Priming for enhanced MeJA-responsive gene expression in Arabidopsis plants expressing Pseudomonas fluorescens WCS417r-mediated ISR_genome

Slide: Pieterse_2-1_Control-0h_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_2-1_Control-0h_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_2-1_Control-0h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 0 hours (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolLeaves were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77
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:0.398615
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:
RawQ2.40
NoiseAvg:3.29,Stdev:0.18,Max:3.8,Min:2.8
BackgroundAvg:53.05,Stdev:1.22,Max:57.1,Min:50.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.398615
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-2_Control-1h_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_2-2_Control-1h_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_2-2_Control-1h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 1 hour (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolLeaves were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77
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:0.456313
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:
RawQ2.35
NoiseAvg:3.16,Stdev:0.09,Max:3.4,Min:2.9
BackgroundAvg:50.62,Stdev:1.00,Max:53.4,Min:48.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.456313
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-3_Control-3h_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_2-3_Control-3h_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_2-3_Control-3h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 3 hours (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolLeaves were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77
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:0.461753
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:
RawQ2.28
NoiseAvg:3.20,Stdev:0.25,Max:3.9,Min:2.4
BackgroundAvg:48.24,Stdev:1.24,Max:50.7,Min:42.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.461753
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-4_Control-6h_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_2-4_Control-6h_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_2-4_Control-6h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 6 hours (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolLeaves were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77
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:0.372286
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:
RawQ2.25
NoiseAvg:3.11,Stdev:0.11,Max:3.4,Min:2.9
BackgroundAvg:48.80,Stdev:0.78,Max:50.0,Min:46.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.372286
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-5_ISR-0h_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_2-5_ISR-0h_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_2-5_ISR-0h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 0 hours (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. For induction of ISR, non-pathogenic Pseudomonas fluorescens WCS417r bacteria were used mized through the soil. For control plants an equal volume of 10 mM MgSO4 was mixed through the soil. WCS417r was grown for 24 hours at 28°C on King's medium B agar plates containing the appropriate antibiotics as described previously (Pieterse et al. 1996: Plant Cell 8: 1225-1237)). Bacteria were collected and resuspended in 10 mM MgSO4 to a density of 10E9 cfu/mL (OD660=1.0) before being mixed through soil. WCS417r bacteria were mixed through the soil to a final density of 5.10E7 cfu/g soil (50 mL of 10E9 cfu/mL mixed through each kg of soil). Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolPlants were grown in soil containing ISR-inducing Pseudomonas fluorescens WCS417r bacteria to a final density of 5x10E7 cfu per gram of soil. When 5 weeks old, plants were dipped were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77.
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:0.260973
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:
RawQ34.46
NoiseAvg:337.48,Stdev:18.84,Max:405.1,Min:284.9
BackgroundAvg:2271.45,Stdev:139.89,Max:2805.9,Min:1933.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.260973
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-6_ISR-1h_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_2-6_ISR-1h_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_2-6_ISR-1h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 1 hour (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. For induction of ISR, non-pathogenic Pseudomonas fluorescens WCS417r bacteria were used mized through the soil. For control plants an equal volume of 10 mM MgSO4 was mixed through the soil. WCS417r was grown for 24 hours at 28°C on King's medium B agar plates containing the appropriate antibiotics as described previously (Pieterse et al. 1996: Plant Cell 8: 1225-1237)). Bacteria were collected and resuspended in 10 mM MgSO4 to a density of 10E9 cfu/mL (OD660=1.0) before being mixed through soil. WCS417r bacteria were mixed through the soil to a final density of 5.10E7 cfu/g soil (50 mL of 10E9 cfu/mL mixed through each kg of soil). Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolPlants were grown in soil containing ISR-inducing Pseudomonas fluorescens WCS417r bacteria to a final density of 5x10E7 cfu per gram of soil. When 5 weeks old, plants were dipped were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77.
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:0.371281
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:
RawQ2.41
NoiseAvg:3.16,Stdev:0.06,Max:3.5,Min:3.0
BackgroundAvg:53.50,Stdev:0.55,Max:55.0,Min:51.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.371281
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-7_ISR-3h_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_2-7_ISR-3h_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_2-7_ISR-3h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 3 hours (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. For induction of ISR, non-pathogenic Pseudomonas fluorescens WCS417r bacteria were used mized through the soil. For control plants an equal volume of 10 mM MgSO4 was mixed through the soil. WCS417r was grown for 24 hours at 28°C on King's medium B agar plates containing the appropriate antibiotics as described previously (Pieterse et al. 1996: Plant Cell 8: 1225-1237)). Bacteria were collected and resuspended in 10 mM MgSO4 to a density of 10E9 cfu/mL (OD660=1.0) before being mixed through soil. WCS417r bacteria were mixed through the soil to a final density of 5.10E7 cfu/g soil (50 mL of 10E9 cfu/mL mixed through each kg of soil). Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolPlants were grown in soil containing ISR-inducing Pseudomonas fluorescens WCS417r bacteria to a final density of 5x10E7 cfu per gram of soil. When 5 weeks old, plants were dipped were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77.
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:0.337534
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:
RawQ2.25
NoiseAvg:3.32,Stdev:0.09,Max:3.6,Min:3.0
BackgroundAvg:50.31,Stdev:0.50,Max:51.8,Min:49.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.337534
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Pieterse_2-8_ISR-6h_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_2-8_ISR-6h_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_2-8_ISR-6h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Age: 6 hours (The time course started immediately after MeJA treatment)
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 with a 24-h time interval. For induction of ISR, non-pathogenic Pseudomonas fluorescens WCS417r bacteria were used mized through the soil. For control plants an equal volume of 10 mM MgSO4 was mixed through the soil. WCS417r was grown for 24 hours at 28°C on King's medium B agar plates containing the appropriate antibiotics as described previously (Pieterse et al. 1996: Plant Cell 8: 1225-1237)). Bacteria were collected and resuspended in 10 mM MgSO4 to a density of 10E9 cfu/mL (OD660=1.0) before being mixed through soil. WCS417r bacteria were mixed through the soil to a final density of 5.10E7 cfu/g soil (50 mL of 10E9 cfu/mL mixed through each kg of soil). Plants were cultivated in a growth chamber with a 9-h day (200 uE/m2/sec at 24oC) and 15-h night (20oC) cycle at 70 percent 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.
LocationGrowth chamber
Growth substrateCommercial soil
SoilWe used a sand/potting soil mixture (5 volumes of river sand and 12 volumes of potting soil).
SterilizationThe sand/potting soil mixuture was autoclaved twice at 121 oC for 20 with a 24-h time interval
WateringPlants were watered every other day and received half-strength Hoagland nutrient solution containing 10 uM Sequestreen once a week
Average humidity70%
Average temperature21.5oC
Growth mediumHoagland's No.2 basal salt mixture
Developmental Stage:
Developmental stage(Source: Boye's Key - Paradigm Genetics)3.90
Tissue: whole rosette
in vivo Treatment:
Treatment protocolPlants were grown in soil containing ISR-inducing Pseudomonas fluorescens WCS417r bacteria to a final density of 5x10E7 cfu per gram of soil. When 5 weeks old, plants were dipped were dipped in a solution containing 50 µM MeJA and 0.015% of Silwet L-77.
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:0.48556
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.75
NoiseAvg:2.46,Stdev:0.08,Max:2.8,Min:2.2
BackgroundAvg:37.82,Stdev:0.35,Max:39.2,Min:37.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.485560
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team