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Experiment: Systemic response to avirulent bacterial infection

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-403

In the absence of adaptive immunity displayed by animals, plants respond locally to biotic challenge via inducible basal defense networks activated through recognition and response toconserved pathogen associated molecular patterns (PAMPs). In addition, immunity can be induced in tissues remote from infection sites via systemic acquired resistance (SAR), initiated following gene-for-gene recognition between plant resistance proteins and microbial effectors.The nature of the mobile signal and remotely activated networks responsible for establishing SAR remain unclear.

Here we show that despite the absence of PAMP contact, systemically responding leaves rapidly activate a SAR transcriptional signature with strong similarity to local basal defense. Jasmonates have previously been implicated in systemic signalling in response to wounding and plant herbivory but not SAR. We present several lines of evidence that suggest jasmonates may also be central to SAR. Jasmonic acid (JA) rapidly accumulates in phloem exudates of leaves challenged with an avirulent strain of Pseudomonas syringae. In systemically responding leaves transcripts associated with jasmonate biosynthesis are upregulated and JA increases transiently. SAR can be mimicked by foliar JA application and is abrogated in mutants impaired in jasmonate synthesis or response.

We conclude that, jasmonate signalling appears to mediate long-distance information transmission. Moreover, the systemic transcriptional response shares extraordinary overlap with local herbivory and wounding responses, indicating that jasmonates may be central to an evolutionarily conserved signalling network, which decodes multiple abiotic and biotic stress signals.

About the Experimenter

Name:Dr William Truman
Head of Lab Name:Prof Murray Grant
Lab:
Institute: University of Exeter
Address:School of Biosciences
Geoffrey Pope Building
Stocker Road
Exeter
Devon
Postcode: EX4 4QD
Country: UK
 
Telephone Number: +44 (0)1392 263789
Fax Number: +44 (0)1392 263434

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

About this Experiment

Experiment Type: pathogenicity_design
Number of Slides:9
 
Experimental Parameters:
parameterinfect
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Truman: Systemic response to avirulent bacterial infection_genome

Slide: Truman_1-1_Pst-DC3000-4hpi_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 ("Truman_1-1_Pst-DC3000-4hpi_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: Truman_1-1_Pst-DC3000-4hpi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with virulent Pseudomonas syringae pv tomato DC3000. DC3000 cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000 containing the empty vector plasmid pVSP61 (Innes et al., 1993) Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.653835
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.29
NoiseAvg:5.13,Stdev:0.41,Max:6.2,Min:4.2
BackgroundAvg:84.93,Stdev:2.92,Max:93.6,Min:78.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.653835
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-2_Pst-DC3000(hrpA)-4hpi_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 ("Truman_1-2_Pst-DC3000(hrpA)-4hpi_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: Truman_1-2_Pst-DC3000(hrpA)-4hpi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with Pseudomonas syringae pv tomato DC3000::hrpA- mutant. DC3000::hrpA- cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000::hrpA- containing the empty vector plasmid pVSP61 (Innes et al., 1993) Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.829904
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.95
NoiseAvg:7.29,Stdev:0.69,Max:9.0,Min:5.8
BackgroundAvg:109.33,Stdev:6.17,Max:126.4,Min:96.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.829904
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-3_Pst-DC3000(avrRpm1)-4hpi_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 ("Truman_1-3_Pst-DC3000(avrRpm1)-4hpi_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: Truman_1-3_Pst-DC3000(avrRpm1)-4hpi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with avirulent Pseudomonas syringae pv tomato DC3000(avrRpm1). DC3000(avrRpm1) cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000 containing the vector plasmid pVSP61 (Innes et al., 1993) carrying the avirulence gene avrRpm1. Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.579079
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.80
NoiseAvg:6.18,Stdev:0.79,Max:10.8,Min:5.2
BackgroundAvg:94.43,Stdev:3.73,Max:105.5,Min:88.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.579079
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-4_Pst-DC3000-4hpi_Rep2_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Truman_1-4_Pst-DC3000-4hpi_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Truman_1-4_Pst-DC3000-4hpi_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with virulent Pseudomonas syringae pv tomato DC3000. DC3000 cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000 containing the empty vector plasmid pVSP61 (Innes et al., 1993) Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:1.185623
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:
RawQ2.90
NoiseAvg:3.47,Stdev:0.29,Max:4.3,Min:2.7
BackgroundAvg:70.58,Stdev:3.29,Max:81.1,Min:64.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.185623
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-5_Pst-DC3000(hrpA)-4hpi_Rep2_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Truman_1-5_Pst-DC3000(hrpA)-4hpi_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Truman_1-5_Pst-DC3000(hrpA)-4hpi_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with Pseudomonas syringae pv tomato DC3000::hrpA- mutant. DC3000::hrpA- cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000::hrpA- containing the empty vector plasmid pVSP61 (Innes et al., 1993) Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.674466
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.95
NoiseAvg:7.46,Stdev:0.59,Max:8.8,Min:5.7
BackgroundAvg:117.56,Stdev:5.78,Max:135.3,Min:104.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.674466
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-6_Pst-DC3000(avrRpm1)-4hpi_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Truman_1-6_Pst-DC3000(avrRpm1)-4hpi_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Truman_1-6_Pst-DC3000(avrRpm1)-4hpi_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with avirulent Pseudomonas syringae pv tomato DC3000(avrRpm1). DC3000(avrRpm1) cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000 containing the vector plasmid pVSP61 (Innes et al., 1993) carrying the avirulence gene avrRpm1. Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.651384
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.50
NoiseAvg:5.18,Stdev:0.60,Max:6.8,Min:3.9
BackgroundAvg:88.11,Stdev:4.50,Max:98.6,Min:76.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.651384
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-7_Pst-DC3000-4hpi_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Truman_1-7_Pst-DC3000-4hpi_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Truman_1-7_Pst-DC3000-4hpi_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with virulent Pseudomonas syringae pv tomato DC3000. DC3000 cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000 containing the empty vector plasmid pVSP61 (Innes et al., 1993) Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.667357
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.07
NoiseAvg:4.35,Stdev:0.28,Max:5.1,Min:3.7
BackgroundAvg:79.77,Stdev:2.28,Max:86.6,Min:74.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.667357
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-8_Pst-DC3000(hrpA)-4hpi_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Truman_1-8_Pst-DC3000(hrpA)-4hpi_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Truman_1-8_Pst-DC3000(hrpA)-4hpi_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with Pseudomonas syringae pv tomato DC3000::hrpA- mutant. DC3000::hrpA- cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000::hrpA- containing the empty vector plasmid pVSP61 (Innes et al., 1993) Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:2.246051
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.42
NoiseAvg:2.51,Stdev:0.14,Max:2.8,Min:2.2
BackgroundAvg:58.74,Stdev:1.43,Max:63.0,Min:54.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.246051
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Truman_1-9_Pst-DC3000(avrRpm1)-4hpi_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Truman_1-9_Pst-DC3000(avrRpm1)-4hpi_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Truman_1-9_Pst-DC3000(avrRpm1)-4hpi_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
LocationGrowth Room
Growth substrate(Source: Commercial soil)A 4:1 mixture of Levingtons F2 compost (Fisons, Ipswich, UK) and vermiculite
stratification2 days in fridge at 4oCA
Growth protocolArabidopsis thaliana genotypes were sown in Levingtons F2 compost (Fisons, Ipswich, UK) and vernalized for 2 days at 4oC. Plants were grown under short day conditions in a controlled environment chamber (12 h light, 100?150 uE at 22oC day, 20oC night) for 5 weeks before use
Developmental Stage:
developmental-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.90
Tissue: whole leaves from the inner rosette (systemic/ uninoculated leaves)
in vivo Treatment:
treatmentLeaves were inoculated with avirulent Pseudomonas syringae pv tomato DC3000(avrRpm1). DC3000(avrRpm1) cultures were grown overnight in 10 ml of Kings B solution (King et al., 1954) supplemented with antibiotics. Plants were inoculated with DC3000 containing the vector plasmid pVSP61 (Innes et al., 1993) carrying the avirulence gene avrRpm1. Antibiotics rifampicin(50 lg ml)1) and kanamycin (25 lg ml)1) were used for selection of the bacterium and plasmid, respectively. Overnight cultures were washed once in 10 mM MgCl2 and final cell densities were adjusted to OD600 0.2. Both sides of a leaf were infiltrated on their abaxial surface with a needleless 1ml syringe. At least 5 leaves per plant were inoculated and 16 plants were pooled to make this sample.
Additional Organism Information:
sample description Systemic, uninfected leaves from the inner rosette of developing, not yet fully expanded, leaves were harvested 4hpi
Separation Technique: The systemic leaves were removed with dissection scissors
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.752644
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.15
NoiseAvg:4.22,Stdev:0.16,Max:4.8,Min:3.7
BackgroundAvg:79.66,Stdev:1.79,Max:85.9,Min:76.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.752644
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team