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Experiment: Incompatible interaction Plasmodiophora-Arabidopsis

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-400

A monogenetic inherited resistance phenotype to Plasmodiophora brassicae has been found in Arabidopsis ecotype Tsu-0. The dominantly inherited RPB1 gene mediates a hypersensitive-like response reaction. Inoculated roots of the resistant line will be compared to control roots at different time points after inoculation in order to get insights into the incompatible interaction. The corresponding dataset has been published (Siemens et al. 2006, MPMI). Furthermore, crossing experiments of the line Tsu-0 with mutant lines of salicylic acid, jasmonic acid and ethylene revealed no influence of these hormones in the resistance reaction. Furthermore, mutations of sgt1b and rar1 showed no influence on RPB1 mediated resistance, but a mutation of sgt1a (At4g23570) reveals to be epistatic to RPB1, indicating to protein ubiquitination and protein degradation as important part of clubroot resistance mechanism. The RPB1 locus has been localized to a region on chromosome 1 with 14 coding sequences according to the sequence of ecotype Columbia. One of these genes is polymorphic between resistant and susceptible ecotypes and encodes for a protein interacting with a SCF-complex. Therefor this gene is probably identical to RPB1.

About the Experimenter

Name:Dr. Johannes Siemens
Head of Lab Name:Dr. Johannes Siemens
Lab:
Institute: Technical University Dresden
Address:Technical University Dresden
Dept. Biology
Molecular Biotechnology
Dresden
Postcode: 01062
Country: Germany
 
Telephone Number: +49 351 463 36789
Fax Number: +49 351 463 37749

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:
Number of Slides:6
 
Experimental Parameters:
exp-factorsinfect
Quality Control Measures Taken:
References:
 
Other Information:
other factorsInfected with Plasmodiophora brassicae

Slides in this Experiment

Hybridisation Set: Siemens: Incompatible interaction Plasmodiophora-Arabidopsis_genome

Slide: Siemens_1-1_Tsu10dpi_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 ("Siemens_1-1_Tsu10dpi_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: Siemens_1-1_Tsu10dpi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Tsu-0
Stock Code: N1564
Age: 24 dpg, 10 dpi
Growth Conditions:
StratificationSeeds were placed on wet filter paper and stored in the refrigerator for 4 days. Afterwards the seeds were put on top of wet soil with a pair of tweezers.
ProtocolPlants were grown in soil under controlled environment (21°C, 16 h light, 100 µmol photons/s/m2).
ReferenceFuchs and Sacristan (1996) MPMI 9:91-97, 1996 Siemens et al. (2002) J.Phytopathology 150:592-605, 2002
Soil ConstituentsThe plants were grown in a soil mixture of 4 parts commercial soil (Einheitserde P, Einheitserde Werkverband, Sinntal-Jossa, Germany) and 1 part gravel (0.8-1.2 mm). The soil "Einheitserde P" is a mixture of peat and clay at pH 6.0 and contains 180 mg/L N, 200 mg/L P and 250 mg/L K.
Substrate Sterilising ProcedureThe soil mixture has been autoclaved just before starting the experiment.
Plant Spacing2,5 cm distance
waterevery two days watering
Temperature21 °C average, 21 °C day, 21 °C night
Humidity80 % average, 80 % day, 70 % night
Medium
Lighting(Source: High pressure sodium and metal halide manufactured by Osram. Intensity: 100µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.50
Tissue: roots
Additional Organism Information:
Sample DescriptionThe plants were dug up 28 days after germination and soil was removed from the roots with the help of brushes.
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:2.485287
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:
RawQ1.98
NoiseAvg:2.31,Stdev:0.23,Max:3.7,Min:1.8
BackgroundAvg:52.75,Stdev:3.17,Max:55.6,Min:32.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.485287
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Siemens_1-2_Tsu10dpi_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Siemens_1-2_Tsu10dpi_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: Siemens_1-2_Tsu10dpi_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Tsu-0
Stock Code: N1564
Age: 24 dpg, 10 dpi
Growth Conditions:
StratificationSeeds were placed on wet filter paper and stored in the refrigerator for 4 days. Afterwards the seeds were put on top of wet soil with a pair of tweezers.
ProtocolPlants were grown in soil under controlled environment (21°C, 16 h light, 100 µmol photons/s/m2).
ReferenceFuchs and Sacristan (1996) MPMI 9:91-97, 1996 Siemens et al. (2002) J.Phytopathology 150:592-605, 2002
Soil ConstituentsThe plants were grown in a soil mixture of 4 parts commercial soil (Einheitserde P, Einheitserde Werkverband, Sinntal-Jossa, Germany) and 1 part gravel (0.8-1.2 mm). The soil "Einheitserde P" is a mixture of peat and clay at pH 6.0 and contains 180 mg/L N, 200 mg/L P and 250 mg/L K.
Substrate Sterilising ProcedureThe soil mixture has been autoclaved just before starting the experiment.
Plant Spacing2,5 cm distance
waterevery two days watering
Temperature21 °C average, 21 °C day, 21 °C night
Humidity80 % average, 80 % day, 70 % night
Medium
Lighting(Source: High pressure sodium and metal halide manufactured by Osram. Intensity: 100µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.50
Tissue: roots
Additional Organism Information:
Sample DescriptionThe plants were dug up 28 days after germination and soil was removed from the roots with the help of brushes.
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:2.61955
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:
RawQ2.10
NoiseAvg:2.19,Stdev:0.04,Max:2.4,Min:2.1
BackgroundAvg:55.75,Stdev:0.49,Max:57.1,Min:54.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.619550
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Siemens_1-3_Tsu14dpi_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 ("Siemens_1-3_Tsu14dpi_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: Siemens_1-3_Tsu14dpi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Tsu-0
Stock Code: N1564
Age: 28 dpg
Growth Conditions:
StratificationSeeds were placed on wet filter paper and stored in the refrigerator for 4 days. Afterwards the seeds were put on the top of wet soil with a pair of tweezers.
ProtocolPlants were grown in soil under controlled environment (21°C, 16h light, 100 µmol photons/s/m2)
ReferenceFuchs and Sacristan (1996) MPMI 9: 91-97, 1996 Siemens et al. (2002) J. Phytopathology 150: 592-605, 2002
Soil ConstituentsThe plants were grown in a soil mixture of 4 parts commercial soil (Einheitserde P, Einheitserde Werkverband, Sinntal-Jossa, Germany) and 1 part gravel (0.8-1.2 mm size) The soil "Einheitserde P" is a mixture of peat and clay at pH 6.0 and contains 180 mg/l N, 200 mg/l P and 250 mg/l K.
Substrate Sterilising ProcedureThe soil mixture has been autoclaved just before starting the experiment
Plant Spacing2,5 cm distance
waterevery 2 days watering
Temperature21 °C average, 21 °C day, 21 °C night
Humidity80 % average, 80 % day, 70 % night
Medium
Lighting(Source: High pressure sodium and metal halide manufactured by Osram. Intensity: 100µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.50
Tissue: roots
in vivo Treatment: Fourteen day old plants were inoculated by injecting the soil around the plant with 2 ml of a resting spore suspension with a concentration of 10 to 6 spores/ml of the pathogen Plasmodiophora brassicae isolate "e3".
Additional Organism Information:
Sample DescriptionThe plants were dug up 14 days after inoculation (28 days after germination) by soil was removed from the roots with the help of brushes.
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:1.323731
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.19
NoiseAvg:2.74,Stdev:0.08,Max:3.0,Min:2.5
BackgroundAvg:61.31,Stdev:0.75,Max:63.0,Min:58.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.323731
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Siemens_1-4_Tsu24dpg_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 ("Siemens_1-4_Tsu24dpg_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: Siemens_1-4_Tsu24dpg_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Tsu-0
Stock Code: N1564
Age: 24 dpg
Growth Conditions:
StratificationSeeds were placed on wet filter paper and stored in the refrigerator for 4 days. Afterwards the seeds were put on top of wet soil with a pair of tweezers.
ProtocolPlants were grown in soil under controlled environment (21°C, 16 h light, 100 µmol photons/s/m2).
ReferenceFuchs and Sacristan (1996) MPMI 9:91-97, 1996 Siemens et al. (2002) J.Phytopathology 150:592-605, 2002
Soil ConstituentsThe plants were grown in a soil mixture of 4 parts commercial soil (Einheitserde P, Einheitserde Werkverband, Sinntal-Jossa, Germany) and 1 part gravel (0.8-1.2 mm). The soil "Einheitserde P" is a mixture of peat and clay at pH 6.0 and contains 180 mg/L N, 200 mg/L P and 250 mg/L K.
Substrate Sterilising ProcedureThe soil mixture has been autoclaved just before starting the experiment.
Plant Spacing2,5 cm distance
waterevery two days watering
Temperature21 °C average, 21 °C day, 21 °C night
Humidity80 % average, 80 % day, 70 % night
Medium
Lighting(Source: High pressure sodium and metal halide manufactured by Osram. Intensity: 100µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.50
Tissue: roots
Additional Organism Information:
Sample DescriptionThe plants were dug up 28 days after germination and soil was removed from the roots with the help of brushes.
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:3.461802
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:2.27,Stdev:0.06,Max:2.4,Min:2.1
BackgroundAvg:61.39,Stdev:0.60,Max:63.1,Min:59.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF3.461802
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Siemens_1-5_Tsu28dpg_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 ("Siemens_1-5_Tsu28dpg_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: Siemens_1-5_Tsu28dpg_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Tsu-0
Stock Code: N1564
Age: 28 dpg
Growth Conditions:
StratificationSeeds were placed on wet filter paper and stored in the refrigerator for 4 days. Afterwards the seeds were put on the top of wet soil with a pair of tweezers.
ProtocolPlants were grown in soil under controlled environment (21°C, 16h light, 100 µmol photons/s/m2)
ReferenceFuchs and Sacristan (1996) MPMI 9: 91-97, 1996 Siemens et al. (2002) J. Phytopathology 150: 592-605, 2002
Soil ConstituentsThe plants were grown in a soil mixture of 4 parts commercial soil (Einheitserde P, Einheitserde Werkverband, Sinntal-Jossa, Germany) and 1 part gravel (0.8-1.2 mm size) The soil "Einheitserde P" is a mixture of peat and clay at pH 6.0 and contains 180 mg/l N, 200 mg/l P and 250 mg/l K.
Substrate Sterilising ProcedureThe soil mixture has been autoclaved just before starting the experiment
Plant Spacing2,5 cm distance
waterevery 2 days watering
Temperature21 °C average, 21 °C day, 21 °C night
Humidity80 % average, 80 % day, 70 % night
Medium
Lighting(Source: High pressure sodium and metal halide manufactured by Osram. Intensity: 100µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.50
Tissue: roots
Additional Organism Information:
Sample DescriptionThe plants were dug up 28 days after germination and soil was removed from the roots with the help of brushes.
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:2.163872
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:
RawQ1.94
NoiseAvg:2.24,Stdev:0.07,Max:2.4,Min:2.0
BackgroundAvg:53.54,Stdev:0.42,Max:54.3,Min:51.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.163872
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Siemens_1-6_Tsu24dpg_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Siemens_1-6_Tsu24dpg_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: Siemens_1-6_Tsu24dpg_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Tsu-0
Stock Code: N1564
Age: 24
Growth Conditions:
StratificationSeeds were put on wet filter paper for 3 days at 4°C before they were placed on the top of soil.
ProtocolPlants were grown in soil under 16 h light/8h darkness (100 µEinstein).
ReferenceSiemens et al. 2002, Siemens et al. 2006
Soil ConstituentsEinheitserde P mixed with Quarz sand (3 part soil and 1 part sand)
Substrate Sterilising Procedureautoclaved
Plant Spacing2.5 cm
Temperature21 °C average, 21 °C day, 21 °C night
Humidity80% % average, 85 % day, 80 % night
Medium
Lighting(Source: Cool white fluorescent manufactured by CLF analytische laborgeräte GmbH. Intensity: 100µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.20
Tissue: root
Additional Organism Information:
Sample DescriptionPlants were grown in soil for 24 days
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:2.962356
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:
RawQ1.77
NoiseAvg:1.78,Stdev:0.08,Max:2.1,Min:1.6
BackgroundAvg:46.33,Stdev:0.37,Max:47.2,Min:45.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.962356
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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