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Experiment: Expression patterns of genes induced by sugar accumulation during early leaf senescence

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-136

The aim of our experiment is to analyse the expression pattern of genes induced by sugar accumulation during early leaf senescence.We have recently shown that senescence is induced by glucose in combination with low, but not with high nitrogen supply (Wingler et al. (2004), New Phytol, in press).

We would like to use Affymetrix analysis to compare senescence-specific gene expression in plants grown on

(i) low nitrogen without glucose (LNM),

(ii) low nitrogen with 2% glucose (LNG) and

(iii) high nitrogen with 2% glucose (HNG).

Comparison of (i) and (ii) will allow us to determine the effect of glucose, while comparison of (ii) and (iii) will reveal effects of nitrogen supply.

For this experiment, plants will be harvested on day 30, when first signs of senescence become visible on the LNG medium. All samples will be taken around mid-day. For each sample, 12 plants from 3 different petri dishes will be pooled. We are planning to analyse 3 independent samples per treatment, i.e. 9 samples in total. RNA will be extracted from whole leaf rosettes (without roots and inflorescences) using the RNeasy Plant Kit (Qiagen).

At a later stage, we are also intending to compare gene expression in wild-type plants with expression in the abi5-1 mutant (Ws-2 background), which shows a delay in sugar-induced senescence (Wingler, Astrid, Marès, Magali & Pourtau, Nathalie (2004)

Spatial patterns and metabolic regulation of photosynthetic parameters during leaf senescence.

New Phytologist 161 (3), 781-789). We have therefore chosen to use Ws-2 for the experiment proposed here. The experiment with abi5-1 will be conducted once we have obtained data on treatment-specific effects in Ws-2.

About the Experimenter

Name:Dr Nathalie Pourtau
Head of Lab Name:Dr Astrid Wingler
Lab:
Institute: University College London
Address:University College London, Biology Department, Gower Street, LONDON.
Postcode: WC 1E 6BT
Country: UK
 
Telephone Number: 020 7679 7268
Fax Number: 020 7679 7096

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:9
 
Experimental Parameters:
parameternutrients
Quality Control Measures Taken:
no-plants-pooled12
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Pourtau_genome

Slide: Pourtau_1-9_highN-glu_Rep3_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 ("Pourtau_1-9_highN-glu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Pourtau_1-9_highN-glu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 30 mM nitrogen (10.3mM NH4+ and 19.7mM NO3-). For sugar treatments, 2% of glucose will be added to the corresponding medium.
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.396126419306
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: Pourtau_1-6_lowN-glu_Rep3_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 ("Pourtau_1-6_lowN-glu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Pourtau_1-6_lowN-glu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 4.7mM (NO3- only). For sugar treatments, 2% of glucose will be added to the corresponding medium.
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.36847358942
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: Pourtau_1-3_lowN_Rep3_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 ("Pourtau_1-3_lowN_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Pourtau_1-3_lowN_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 4.7mM (NO3- only).
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.375910311937
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: Pourtau_1-8_highN-glu_Rep2_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Pourtau_1-8_highN-glu_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: Pourtau_1-8_highN-glu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 30 mM nitrogen (10.3mM NH4+ and 19.7mM NO3-). For sugar treatments, 2% of glucose will be added to the corresponding medium.
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-09-09

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:0.530739068985
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: Pourtau_1-5_lowN-glu_Rep2_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Pourtau_1-5_lowN-glu_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: Pourtau_1-5_lowN-glu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 4.7mM (NO3- only). For sugar treatments, 2% of glucose will be added to the corresponding medium.
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.41105863452
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: Pourtau_1-2_lowN_Rep2_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Pourtau_1-2_lowN_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: Pourtau_1-2_lowN_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 4.7mM (NO3- only).
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.414479404688
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: Pourtau_1-7_highN-glu_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 ("Pourtau_1-7_highN-glu_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: Pourtau_1-7_highN-glu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 30 mM nitrogen (10.3mM NH4+ and 19.7mM NO3-). For sugar treatments, 2% of glucose will be added to the corresponding medium.
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.405542582273
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: Pourtau_1-4_lowN-glu_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 ("Pourtau_1-4_lowN-glu_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: Pourtau_1-4_lowN-glu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 4.7mM (NO3- only). For sugar treatments, 2% of glucose will be added to the corresponding medium.
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.311314165592
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: Pourtau_1-1_lowN_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 ("Pourtau_1-1_lowN_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: Pourtau_1-1_lowN_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ws-2
Stock Code: N1601
Growth Conditions:
Humidity50% relative humidity within the growth cabinet
LightPlants will be grown in a controlled-environment cabinet in vertical orientation at a photon flux density of 100umol.m-2.s-1 for 16 hours per day.
MediumPlants will be grown on 1% agar media.
Temperature22oC during the day and 18oC during the night.
Locationgrowth cabinet
HarvestingPlants will be harvested under illumination at mid-day.
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)6.00-first_flower_bud_open
Tissue: whole leaf rosettes without the roots and inflorescences
Diseased: Normal
in vivo Treatment: Half-strength Murashige and Skoog medium containing 4.7mM (NO3- only).
Additional Organism Information:
tissueWe are going to use whole leaf rosettes without the roots and inflorescences (to avoid treatment-dependent effects on flowers-specific gene expression).
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2004-08-26

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:0.941013336182
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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