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Experiment: UV-B Responses in Light Grown Plants: Similarities to Biotic Stress

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-56

UV-B (280-320 nm) exposure causes serious damage in plants, limiting their growth and survival, effects that are partly counteracted by repair mechanisms active in plants receiving accompanying visible radiation. Though no particular UV-B receptor has been identified to date, there is strong evidence to indicate that certain aspects of UV-B perception are receptor-mediated. Investigations of down-stream signalling events have thus far indicated broad similarities to pathogen-induced defence responses in plants. In order to identify genes in Arabidopsis that may be up- or down- regulated specifically in response to UV-B exposure and compare them to genes whose expression is altered in plants challenged by an avirulent isolate of Peronospora parasitica (downy mildew), we propose to analyse the transcriptional profiles for the following treatments:

1. UV-B Responses

"A-1" Columbia (Col-0) exposed to supplementary UV-B/UV-A* with a background of low photosynthetically active radiation (PAR of 20 micromol m-2 s-1) for 1.5 photoperiods (photoperiod = 12h). [UV-B treatment]

"A-2" Col-0 exposed to supplementary UV-A and low PAR for 1.5 photoperiods [control for UV-B treatment]

"A-3" Col-0 exposed to visible light only (low PAR) (no UV) for 1.5 photoperiods [control for UV effects in general].* There are no pure sources of UV-B light available.

2. Pathogen Responses

"A-4" Col-0 spray-inoculated with P. parasitica isolate HIKS-1 (recognised by the R-gene RPP7). After spraying, plants were kept covered in plant propagators and transferred to an 18 degreeC growth chamber. Samples for RNA extraction were taken 72h after inoculation.

"A-5" The viability of spores was also checked by parallel spraying of the susceptible mutant, Col-rpp7. [pathogen treatment]

"A-6" Col-0 mock treated with water, covered and transferred to an 18 degree C growth chamber, 72h prior to sampling. [control for pathogen treatment]

In all experiments, we are using RNA from leaves taken at the same time of day (6 h into the 12 h photoperiod) from 4.5-week old plants grown under 12h photoperiod. All treatments were normalised against PR-1 expression levels to ensure comparability between UV-B and pathogen treatments. Due to the difficulty in distinguishing between local and systemic induced responses in UV-B treated plants, we are using RNA from whole rosettes for both the UV-B and pathogen treatment for better comparability among treatments. The degree of similarity between these two sets of transcriptional changes will complement and help interpret our experimental data on changes in resistance to pathogens in plants pre-treated with UV-B. Moreover, the data set obtained would allow for identification of UV-B specific changes in gene expression including cis-acting UV-B-responsive promoter elements.

About the Experimenter

Name:Miss Julia Brueggemann
Head of Lab Name:Dr Eric Holub
Lab: Horticulture Research International
Institute: University of Warwick
Address:Horticulture Research International
Wellesbourne
Warwickshire
Postcode: CV35 9EF
Country: UK
 
Telephone Number: 01789 470 382
Fax Number: 01789 470 552

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: stimulus_or_stress_design; genetic_modification_design
Number of Slides:6
 
Experimental Parameters:
parameteratmosphere
parameterinfect
parametergene_knock_out
Quality Control Measures Taken:
no-plants-pooled3 plants.
References:
 
Other Information:
ArrayExpress AccessionE-NASC-16

Slides in this Experiment

Hybridisation Set: Brueggemann_genome

Slide: Brueggemann_1-1_Col0-UVB_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 ("Brueggemann_1-1_Col0-UVB_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: Brueggemann_1-1_Col0-UVB_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity70%
Temperature21?C (day) 16?C (night)
Lighting(Source: 150 micromol m-2 s-1)12 hr light: 12 hr dark
MediumCompost
Nutrientsnone
Watertop watering as needed 1-2 times a week
Stratificationno
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: whole above ground rosettes
Diseased: Normal
in vivo Treatment:
treatmentUV-B TREATMENT: UV-B source: Philips TL12 40W tubes covered with 1 layer (0.12mm) of "Diacel" cellulose diacetate UV-B fluence rate (280nm-320nm): 4.5 micromol m-2 s-1 Background photosynthetically active radiation (400nm-700nm): 20 micromol m-2 s-1 Duration: 1.5 photoperiods (samples taken 6 hours into the second day of treatment with supplementary UV-B)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Brueggemann TMS
Method:
ProtocolGround plant tissue was extracted in a mixture of 5 ml Phenol:Chloroform:IAA (25:24:1), 5 ml TNS buffer (4% w/v p-aminoalicylic acid, 1% w/v Triisopropylnaphthalene sulfonic acid sodium salt, 10mM Tris-HCl pH8) and 100 microliter beta-mercaptoethanol. After centrifugation, the aqueous phase was recovered. Nucleic acids were precipitated at -20degreeC in the presence of 0.3 M Sodium Acetate (ph5) and 70% Ethanol, pelleted by centrifugation, and resuspended in water. RNA was precipitated over night on ice in the presence of 2.33M LiCl and subsequently pelleted by centrifugation. The pellet was then washed first with 3M Sodium Acetate pH5 to remove residual DNA and subsequently two more times with 70% Ethanol before resuspending it in DEPC-treated water. The RNA concentration of an aliquot was checked spectrometrically. RNA was stored at -80degreeC.
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: 2003-03-20

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:0.276
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

Slide: Brueggemann_1-2_Col0-UVA_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 ("Brueggemann_1-2_Col0-UVA_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: Brueggemann_1-2_Col0-UVA_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity70%
Temperature21?C (day) 16?C (night)
Lighting(Source: 150 micromol m-2 s-1)12 hr light: 12 hr dark
MediumCompost
Nutrientsnone
Watertop watering as needed 1-2 times a week
Stratificationno
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: whole above ground rosettes
Diseased: Normal
in vivo Treatment:
treatmentUV-A TREATMENT: UV-A source: Philips TL12 40W tubes covered with 1 layer (0.1mm) of "Mylar D" UV-A fluence rate (320nm-400): 3.7 micromol m-2 s-1 Background photosynthetically active radiation (400nm-700nm): 20 micromol m-2 s-1 Duration: 1.5 photoperiods (samples taken 6 hours into the second day of treatment with supplementary UV-B)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Brueggemann TMS
Method:
ProtocolGround plant tissue was extracted in a mixture of 5 ml Phenol:Chloroform:IAA (25:24:1), 5 ml TNS buffer (4% w/v p-aminoalicylic acid, 1% w/v Triisopropylnaphthalene sulfonic acid sodium salt, 10mM Tris-HCl pH8) and 100 microliter beta-mercaptoethanol. After centrifugation, the aqueous phase was recovered. Nucleic acids were precipitated at -20degreeC in the presence of 0.3 M Sodium Acetate (ph5) and 70% Ethanol, pelleted by centrifugation, and resuspended in water. RNA was precipitated over night on ice in the presence of 2.33M LiCl and subsequently pelleted by centrifugation. The pellet was then washed first with 3M Sodium Acetate pH5 to remove residual DNA and subsequently two more times with 70% Ethanol before resuspending it in DEPC-treated water. The RNA concentration of an aliquot was checked spectrometrically. RNA was stored at -80degreeC.
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: 2003-03-20

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:0.334
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

Slide: Brueggemann_1-3_Col0-VIS_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 ("Brueggemann_1-3_Col0-VIS_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: Brueggemann_1-3_Col0-VIS_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1093
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity70%
Temperature21?C (day) 16?C (night)
Lighting(Source: 150 micromol m-2 s-1)12 hr light: 12 hr dark
MediumCompost
Nutrientsnone
Watertop watering as needed 1-2 times a week
Stratificationno
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: whole above ground rosettes
Diseased: Normal
in vivo Treatment:
treatmentVisible Light Only TREATMENT: Background photosynthetically active radiation (400nm-700nm): 20 micromol m-2 s-1 Duration: 1.5 photoperiods (samples taken 6 hours into the second day of treatment with supplementary UV-B)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Brueggemann TMS
Method:
ProtocolGround plant tissue was extracted in a mixture of 5 ml Phenol:Chloroform:IAA (25:24:1), 5 ml TNS buffer (4% w/v p-aminoalicylic acid, 1% w/v Triisopropylnaphthalene sulfonic acid sodium salt, 10mM Tris-HCl pH8) and 100 microliter beta-mercaptoethanol. After centrifugation, the aqueous phase was recovered. Nucleic acids were precipitated at -20degreeC in the presence of 0.3 M Sodium Acetate (ph5) and 70% Ethanol, pelleted by centrifugation, and resuspended in water. RNA was precipitated over night on ice in the presence of 2.33M LiCl and subsequently pelleted by centrifugation. The pellet was then washed first with 3M Sodium Acetate pH5 to remove residual DNA and subsequently two more times with 70% Ethanol before resuspending it in DEPC-treated water. The RNA concentration of an aliquot was checked spectrometrically. RNA was stored at -80degreeC.
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: 2003-03-20

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:0.37
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

Slide: Brueggemann_1-4_Col5-Pparasitica_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 ("Brueggemann_1-4_Col5-Pparasitica_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: Brueggemann_1-4_Col5-Pparasitica_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity70%
Temperature21degreeC (day) 16degreeC (night)
Lighting(Source: 150 micromol m-2 s-1)12 hr light: 12 hr dark
MediumCompost
Nutrientsnone
Watertop watering as needed 1-2 times a week
Stratificationno
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: whole above ground rosettes
Diseased: Normal
in vivo Treatment:
treatment"Peronospora parasitica on resistant plant" TREATMENT: Col-5 plants (resistant host) sprayed with P. parasitica Hiks-1 spores (2*10e4 spores per ml or above) until run-off. After spraying, plants were coverd with propagator lids and transferred to an 18 degree C (day and night) growth room for 3 days before sampling (sampling done 6 hours into the light period)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Brueggemann TMS
Method:
ProtocolGround plant tissue was extracted in a mixture of 5 ml Phenol:Chloroform:IAA (25:24:1), 5 ml TNS buffer (4% w/v p-aminoalicylic acid, 1% w/v Triisopropylnaphthalene sulfonic acid sodium salt, 10mM Tris-HCl pH8) and 100 microliter beta-mercaptoethanol. After centrifugation, the aqueous phase was recovered. Nucleic acids were precipitated at -20degreeC in the presence of 0.3 M Sodium Acetate (ph5) and 70% Ethanol, pelleted by centrifugation, and resuspended in water. RNA was precipitated over night on ice in the presence of 2.33M LiCl and subsequently pelleted by centrifugation. The pellet was then washed first with 3M Sodium Acetate pH5 to remove residual DNA and subsequently two more times with 70% Ethanol before resuspending it in DEPC-treated water. The RNA concentration of an aliquot was checked spectrometrically. RNA was stored at -80degreeC.
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: 2003-03-20

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:0.282
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

Slide: Brueggemann_1-5_Col5-mock-treatment_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 ("Brueggemann_1-5_Col5-mock-treatment_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: Brueggemann_1-5_Col5-mock-treatment_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1688
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity70%
Temperature21degreeC (day) 16degreeC (night)
Lighting(Source: 150 micromol m-2 s-1)12 hr light: 12 hr dark
MediumCompost
Nutrientsnone
Watertop watering as needed 1-2 times a week
Stratificationno
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: whole above ground rosettes
Diseased: Normal
in vivo Treatment:
treatment"Mock" TREATMENT:Plants were sprayed with H2O as a mock treatment.After spraying, plants were coverd with propagator lids and transferred to an 18 degree C (day and night) growth room for 3 days before sampling (sampling done 6 hours into the light period)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Brueggemann TMS
Method:
ProtocolGround plant tissue was extracted in a mixture of 5 ml Phenol:Chloroform:IAA (25:24:1), 5 ml TNS buffer (4% w/v p-aminoalicylic acid, 1% w/v Triisopropylnaphthalene sulfonic acid sodium salt, 10mM Tris-HCl pH8) and 100 microliter beta-mercaptoethanol. After centrifugation, the aqueous phase was recovered. Nucleic acids were precipitated at -20degreeC in the presence of 0.3 M Sodium Acetate (ph5) and 70% Ethanol, pelleted by centrifugation, and resuspended in water. RNA was precipitated over night on ice in the presence of 2.33M LiCl and subsequently pelleted by centrifugation. The pellet was then washed first with 3M Sodium Acetate pH5 to remove residual DNA and subsequently two more times with 70% Ethanol before resuspending it in DEPC-treated water. The RNA concentration of an aliquot was checked spectrometrically. RNA was stored at -80degreeC.
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: 2003-03-21

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:0.236
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

Slide: Brueggemann_1-6_Rpp7-Pparasitica_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 ("Brueggemann_1-6_Rpp7-Pparasitica_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: Brueggemann_1-6_Rpp7-Pparasitica_ATH1
Organism: Arabidopsis thaliana
Alias: rpp7
Stock Code:
Genetic Background: Col-5
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity70%
Temperature21degreeC (day) 16degreeC (night)
Lighting(Source: 150 micromol m-2 s-1)12 hr light: 12 hr dark
MediumCompost
Nutrientsnone
Watertop watering as needed 1-2 times a week
Stratificationno
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Genetic Variation: EMS mutant
Tissue: whole above ground rosettes
Diseased: Normal
in vivo Treatment:
treatment"Peronospora parasitica on susceptible host" TREATMENT: rpp7 Plants (susceptible host) were sprayed with Peronospora parasitica Hiks-1 spores (2*10e4 spores per ml or above) until run-off. After spraying, plants were coverd with propagator lids and transferred to an 18 degree C (day and night) growth room for 3 days before sampling (sampling done 6 hours into the light period)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Brueggemann TMS
Method:
ProtocolGround plant tissue was extracted in a mixture of 5 ml Phenol:Chloroform:IAA (25:24:1), 5 ml TNS buffer (4% w/v p-aminoalicylic acid, 1% w/v Triisopropylnaphthalene sulfonic acid sodium salt, 10mM Tris-HCl pH8) and 100 microliter beta-mercaptoethanol. After centrifugation, the aqueous phase was recovered. Nucleic acids were precipitated at -20degreeC in the presence of 0.3 M Sodium Acetate (ph5) and 70% Ethanol, pelleted by centrifugation, and resuspended in water. RNA was precipitated over night on ice in the presence of 2.33M LiCl and subsequently pelleted by centrifugation. The pellet was then washed first with 3M Sodium Acetate pH5 to remove residual DNA and subsequently two more times with 70% Ethanol before resuspending it in DEPC-treated water. The RNA concentration of an aliquot was checked spectrometrically. RNA was stored at -80degreeC.
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: 2003-03-21

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:0.258
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


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