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Experiment: Identification of Core Genes Regulating Plant Programmed Cell Death (PCD)

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-77

PCD is a highly organised process that is involved in development and in an organisms response to biotic stresses (toxins and avirulent pathogens) and abiotic stresses (such as temperature, water availability, etc.). It is a genetically regulated form of cellular suicide, however in plants the underlying process is poorly understood. Although PCD may occur in response to different stimuli; we believe once it is triggered, one core mechanism is responsible for the cellular demise. It is our aim to identify the elements of this mechanism. We will do this by expanding on the work of a previous user of GARNet's GeneChip microarray facility, Dr. Jodi Swidzinski. She utilised an Arabidopsis cell suspension system; performing microarray analysis on both senescing, and heat shock induced PCD samples. This resulted in data showing that a large number of genes were upregulated (or downregulated) in response to both treatments. We are working under the premise that some of the genes that were similarly regulated under both treatments must be core PCD genes. However because of the large amount of data generated it is difficult to choose appropriate candidate genes (with any degree of confidence that they are core genes) for further study.

We propose to use a third PCD-inducing treatment, involving a mycotoxin, to generate another population of microarray results. The mycotoxin we intend to use is Fumonisin B1 (FB1). This is an extremely potent compound that induces PCD by disrupting ceramide synthesis. We have found Arabidopsis protoplasts to be much more sensitive than cells to the toxin at low concentrations. Protoplasts are treated with 20mM FB1 and RNA is extracted at time points when 0%, 20% and 40% of protoplasts have died. This RNA is then pooled. RNA from methanol treated protoplasts is used as a control. We intend for these RNA samples to be subjected to GeneChip microarray analysis. This would identify genes differentially regulated due to FB1 treatment, which would be interesting in itself. However, by combining this data with that from previous work by Swidzinski (2002) we will be able to decrease the pool of possible core genes further, and increase the chances of selecting an appropriate candidate gene. This will both improve upon existing work and add value to an existing GARNet data set.

About the Experimenter

Name:Mr. Mark Diamond
Head of Lab Name:Dr. Paul McCabe
Lab: Botany Department
Institute: University College Dublin
Address:Botany Department
University College Dublin
Belfield
Postcode: Dublin 4
Country: Ireland
 
Telephone Number: 017162251
Fax Number: 017161153

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

About this Experiment

Experiment Type: compound_treatment_design
Number of Slides:8
 
Experimental Parameters:
parameter compound_based_treatment
Quality Control Measures Taken:
no-plants-pooled3 cultures
biological-replicates4
References:
 
Other Information:
ArrayExpressAccessionE-NASC-22

Slides in this Experiment

Hybridisation Set: Diamond_genome

Slide: Diamond_A-1-Diamo-fum_SLD

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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 ("Diamond_A-1-Diamo-Fum_SRC") 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: Diamond_A-1-Diamo-Fum_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-08-13

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:0.484345
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:
RawQ3.23
NoiseAvg:4.42,Stdev:0.16,Max:4.9,Min:3.8
BackgroundAvg:82.53,Stdev:0.86,Max:85.6,Min:79.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.484345
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-2-Diamo-fum_SLD

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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 ("Diamond_A-2-Diamo-Fum_SRC") 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: Diamond_A-2-Diamo-Fum_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-08-13

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:0.426039
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:
RawQ5.95
NoiseAvg:16.11,Stdev:0.86,Max:17.9,Min:12.5
BackgroundAvg:173.62,Stdev:7.72,Max:198.9,Min:159.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.426039
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-3-Diamo-fum_SLD

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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 ("Diamond_A-3-Diamo-Fum_SRC") 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: Diamond_A-3-Diamo-Fum_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-08-13

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:0.322714
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:
RawQ2.97
NoiseAvg:4.08,Stdev:0.15,Max:4.5,Min:3.4
BackgroundAvg:74.60,Stdev:2.02,Max:79.6,Min:69.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.322714
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-4-Diamo-fum_SLD

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 ("Diamond_A-4-Diamo-Fum_SRC") 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: Diamond_A-4-Diamo-Fum_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-08-13

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:0.369875
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:
RawQ3.39
NoiseAvg:4.98,Stdev:0.15,Max:5.5,Min:4.6
BackgroundAvg:85.23,Stdev:0.92,Max:87.8,Min:82.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.369875
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-1-Diamo-met_SLD

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 ("Diamond_A-1-Diamo-Meth_SRC") 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: Diamond_A-1-Diamo-Meth_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-09-09

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:0.489387
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:
RawQ2.55
NoiseAvg:3.20,Stdev:0.14,Max:3.8,Min:2.8
BackgroundAvg:62.38,Stdev:1.00,Max:67.1,Min:60.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.489387
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-2-Diamo-met_SLD

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 ("Diamond_A-2-Diamo-Meth_SRC") 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: Diamond_A-2-Diamo-Meth_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-09-09

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:0.310634
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:
RawQ3.00
NoiseAvg:4.00,Stdev:0.19,Max:4.6,Min:3.6
BackgroundAvg:78.16,Stdev:2.41,Max:85.9,Min:73.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.310634
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-3-Diamo-met_SLD

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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 ("Diamond_A-3-Diamo-Meth_SRC") 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: Diamond_A-3-Diamo-Meth_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-09-09

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:0.506405
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:
RawQ2.97
NoiseAvg:4.04,Stdev:0.18,Max:4.6,Min:3.6
BackgroundAvg:73.41,Stdev:1.67,Max:78.8,Min:69.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.506405
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Diamond_A-4-Diamo-met_SLD

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 ("Diamond_A-4-Diamo-Meth_SRC") 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: Diamond_A-4-Diamo-Meth_SRC
Organism: Arabidopsis thaliana
Alias: Ler
Stock Code: NW20
Growth Conditions:
LocationCell Culture Incubator
AtmosphereNormal
Humidity
Temperature24C
MediumMurashige and Skoog
TreatmentNAA, Kinetin
NutrientsSucrose
Developmental Stage: Cell culture
Tissue: Cell suspension
Diseased: Normal
in vitro Treatment:
treatmentThree, Six day old dark grown cell cultures were pooled and then protoplasted. Protoplasts were then incubated for 24hrs in a constant temperature room prior to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted from these samples. This RNA was then pooled into one sample.
Other Information:
two-or-more-wts

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: 2003-09-09

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:0.443286
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:4.56,Stdev:0.19,Max:5.2,Min:4.1
BackgroundAvg:83.71,Stdev:1.54,Max:88.6,Min:80.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.443286
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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