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Experiment: Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-485

To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. This data is part of Gutierrez et al. PNAS. 2008 March; 105(12): 4939-4944.

About the Experimenter

Name:Dr. Rodrigo Gutierrez
Head of Lab Name:Dr. Gloria Coruzzi
Lab:
Address:New York University - Department of Biology
Center for Genomics and Systems Biology
100 Washington Square East
8th Floor Brown Building
Postcode: NY 10003
Country: USA
 

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; growth_condition_design;
Number of Slides:8
 
Experimental Parameters:
parametercompound_based_treatment
parameterNutrients
parameterMedia
Quality Control Measures Taken:
References:
ReferenceRodrigo A. Gutiérrez, Trevor L. Stokes, Karen Thum, Xiaodong Xu, Mariana Obertello, Manpreet S. Katari, Milos Tanurdzic, Alexis Dean, Damion C. Nero, C. Robertson McClung, and Gloria M. Coruzzi. "Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1". PNAS 2008 105:4939-4944; published online before print March 14, 2008, doi:10.1073/pnas.0800211105.
 
Other Information:

Slides in this Experiment

Hybridisation Set: Gutierrez: Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1._genome

Slide: Gutierrez_1-8_R03.7.8_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 ("Gutierrez_1-8_R03.7.8_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: Gutierrez_1-8_R03.7.8_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KNO3+ 20 mM NH4NO3 + 1 mM MSX + 10 mM Glu, 2 hr, seedlings, replicate 2
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KNO3, 20 mM NH4NO3, 1 mM MSX, 10 mM Glu
Other Information:
RNAQCNo
RNAReceivedNo

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:5.433427
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:
RawQ1.63
NoiseAvg:1.45,Stdev:0.05,Max:1.6,Min:1.3
BackgroundAvg:43.57,Stdev:1.02,Max:45.2,Min:40.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF5.433427
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-7_R03.7.7_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 ("Gutierrez_1-7_R03.7.7_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: Gutierrez_1-7_R03.7.7_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KNO3+ 20 mM NH4NO3 + 1 mM MSX + 10 mM Glu, 2 hr, seedlings, replicate 1
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KNO3, 20 mM NH4NO3, 1 mM MSX, 10 mM Glu
Other Information:
RNAQCNo
RNAReceivedNo

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:3.804455
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:
RawQ1.90
NoiseAvg:2.08,Stdev:0.06,Max:2.3,Min:1.9
BackgroundAvg:55.82,Stdev:1.33,Max:58.4,Min:53.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF3.804455
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-6_R03.7.6_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Gutierrez_1-6_R03.7.6_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: Gutierrez_1-6_R03.7.6_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KNO3+ 20 mM NH4NO3 + 1 mM MSX, 2 hr, seedlings, replicate 2
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KNO3, 20 mM NH4NO3, 1 mM MSX
Other Information:
RNAQCNo
RNAReceivedNo

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:3.505174
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:
RawQ1.55
NoiseAvg:1.39,Stdev:0.07,Max:1.6,Min:1.2
BackgroundAvg:41.33,Stdev:0.89,Max:43.5,Min:39.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF3.505174
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-5_R03.7.5_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 ("Gutierrez_1-5_R03.7.5_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: Gutierrez_1-5_R03.7.5_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KNO3+ 20 mM NH4NO3 + 1 mM MSX, 2 hr, seedlings, replicate 1
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KNO3, 20 mM NH4NO3, 1 mM MSX
Other Information:
RNAQCNo
RNAReceivedNo

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:5.836345
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:
RawQ1.53
NoiseAvg:1.36,Stdev:0.05,Max:1.5,Min:1.3
BackgroundAvg:41.31,Stdev:0.50,Max:43.2,Min:40.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF5.836345
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-4_R03.7.4_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 ("Gutierrez_1-4_R03.7.4_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: Gutierrez_1-4_R03.7.4_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KNO3+ 20 mM NH4NO3, 2 hr, seedlings, replicate 2
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KNO3, 20 mM NH4NO3
Other Information:
RNAQCNo
RNAReceivedNo

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:4.1999
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:
RawQ2.04
NoiseAvg:2.24,Stdev:0.09,Max:2.4,Min:2.1
BackgroundAvg:62.91,Stdev:1.96,Max:67.1,Min:58.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF4.199900
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-3_R03.7.3_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 ("Gutierrez_1-3_R03.7.3_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: Gutierrez_1-3_R03.7.3_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KNO3+ 20 mM NH4NO3, 2 hr, seedlings, replicate 1
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KNO3, 20 mM NH4NO3
Other Information:
RNAQCNo
RNAReceivedNo

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:5.191806
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:
RawQ1.53
NoiseAvg:1.39,Stdev:0.07,Max:1.5,Min:1.2
BackgroundAvg:41.56,Stdev:1.88,Max:45.6,Min:38.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF5.191806
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-2_R03.7.2_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 ("Gutierrez_1-2_R03.7.2_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: Gutierrez_1-2_R03.7.2_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KCl 2 hr, seedlings, replicate 2
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KCl
Other Information:
RNAQCNo
RNAReceivedNo

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:4.962998
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:
RawQ1.52
NoiseAvg:1.40,Stdev:0.19,Max:2.3,Min:1.2
BackgroundAvg:43.30,Stdev:1.27,Max:49.2,Min:40.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF4.962998
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Gutierrez_1-1_R03.7.1_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 ("Gutierrez_1-1_R03.7.1_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: Gutierrez_1-1_R03.7.1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Sample descriptionWT Col. 20 mM KCl 2 hr, seedlings, replicate 1
Sterilisation protocolSeeds were treated with 50% Commercial bleach, 0.04% Tween-20 for 5 min at room temperature and washed with sterile water 3 times in the hood.
Growth protocolArabidopsis ecotype Columbia plants were grown on basal MS salts with 0.5% (wt/val) sucrose, 0.8% BactoAgar, and 1 mM KNO3 at 22°C under long-day (16/8 hr. light/dark) conditions with 60 mmol photons m-2s-1 light intensity for 14 d.
MediumThe growth medium contained the following components (final concentration): basal MS (H3BO3 6.2 mg/L, Na2MoO4. 2H2O 0.025 mg/L, CaCl2. 2H2O 332.2 mg/L, FeSO4. 7H2O 0.025 mg/L, Na2EDTA 37.26 mg/L, FeSO4. 7H2O 27.8 mg/L, Myo-Inositol 100 mg/L, Na2MoO4 2H2O 0.25 mg/L, MgSO4 180.7 mg/L, MnSO4.H2O 16.9 mg/L, C6H6N2O 0.5 mg/L, KH2PO4 170 mg/L, KI 0.83 mg/L, C8H11NO3.HCl 0.5 mg/L, Thiamine.HCl 0.1 mg/L, ZnSO4.7H2O 8.6 mg/L) supplemented with 1 mM KNO3, 0.5% (wt/vol) sucrose and 0.8 % (wt/vol) BactoAgar at a final pH 5.7.
AverageTemperature22 °C
ReferencePNAS. 2008 March; 105(12): 4939–4944.
Substrate sterilisation methodAutoclaved: 120°C, 1.2 atm, 23 min.
LocationGrowth Chamber
Developmental Stage:
Developmental stage14 days
Tissue: Seedlings
in vivo Treatment:
Treatment20 mM KCl
Other Information:
RNAQCNo
RNAReceivedNo

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:3.352656
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:
RawQ1.57
NoiseAvg:1.51,Stdev:0.05,Max:1.7,Min:1.4
BackgroundAvg:41.09,Stdev:0.97,Max:43.7,Min:38.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF3.352656
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team