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Experiment: Arabidopsis treated with nitrite and nitrate

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-481

Nitrate serves as a potent signal to control gene expression in plants and algae, but little is known about the signaling role of nitrite, the direct product of nitrate reduction. Analysis of several nitrate-induced genes showed that nitrite increases mRNA levels as rapidly as nitrate in nitrogen-starved Arabidopsis (Arabidopsis thaliana) roots. Both nitrite and nitrate induction are apparent at concentrations as low as 100 nm. The response at low nitrite concentrations was not due to contaminating nitrate, which was present at <1% of the nitrite concentration. High levels of ammonium (20 mm) in the growth medium suppressed induction of several genes by nitrate, but had varied effects on the nitrite response. Transcriptome analysis using 250 or 5 microm nitrate or nitrite showed that over one-half of the nitrate-induced genes, which included genes involved in nitrate and ammonium assimilation, energy production, and carbon and nitrogen metabolism responded equivalently to nitrite; however, the nitrite response was more robust and there were many genes that responded specifically to nitrite. Thus, nitrite can serve as a signal as well as if not better than nitrate.

About the Experimenter

Name:Dr. Rongchen Wang
Head of Lab Name:Professor Nigel Crawford
Lab:
Address:University of California, San Diego
Division of Biological Sciences
9500 Gilman Drive, 0116
La Jolla, CA 92093-0116
USA
Postcode: 92093-0116
Country: USA
 
Telephone Number: 18585342519
Fax Number: 18585347108

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:6
 
Experimental Parameters:
parametercompound_based_treatment
parametergene_knock_out
Quality Control Measures Taken:
References:
ReferencePlant Physiology 145:1735-1745 (2007)
 
Other Information:

Slides in this Experiment

Hybridisation Set: Wang: Arabidopsis treated with nitrite and nitrate_genome

Slide: Wang_3-6_WT-Col-250-microM-KNO2-20min-roots_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 ("Wang_3-6_WT-Col-250-microM-KNO2-20min-roots_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: Wang_3-6_WT-Col-250-microM-KNO2-20min-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolArabidopsis seedlings were grown under hydroponic conditions as previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, approximately 100 seedlings were grown at 25°C with continuous light supported on nylon mesh so that roots were submerged in liquid medium and shoots were above the medium. Seedlings were germinated, then grown in 25 mL of liquid medium containing 2.5 mm (NH4)2 succinate as the nitrogen source, 0.5% (w/v) Suc, and other essential macro- and micronutrients for 9 d, then shifted to 100 mL of fresh medium lacking (NH4)2 succinate for 24 h (note that the initial Cl- concentration was over 2 mm). At day 10, treatments were initiated by adding 250 µL solution of KNO2 or KNO3 (treatment) or KCl (control) to the culture to reach the specified concentrations in the medium. Plants were grown for the specified amount of time and then roots were harvested for RNA extraction.
Sterilisation protocolSeeds were treated with 30% Commercial bleach, 0.05% Triton X-100 for 10 min at room temperature and washed with sterile water 5 times in the hood.
Substrate sterilisation protocolIndividual component stock autoclaved separately; FeEDTA filter-sterilized
MediumThe growth medium contained the following components (final concentration): 10 mM potassium phosphate (pH 6.0), 2.5 mM (NH4)2succinate, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNa2EDTA, 0.5% (w/v) Suc, and micro-nutrients (50 µM H3BO3, 12 µM MnSO4, 1 µM ZnCl2, 1 µM CuSO4, and 0.2 µM Na2MoO4). Component solutions were autoclaved (FeNa2EDTA was filter sterilized) separately and added to sterile deionized water before use.
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment250 µM KNO2
Additional Organism Information:
Sample descriptionWT Col., 250 microM KNO2, 20min, roots, replicate 2
Other Information:
ReferencePlant Physiology 145:1735-1745 (2007)

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:0.312402
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:
RawQ2.81
NoiseAvg:3.68,Stdev:0.14,Max:4.0,Min:3.4
BackgroundAvg:77.07,Stdev:0.49,Max:78.8,Min:76.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.312402
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_3-4_WT-Col-250-microM-KCL-20min-roots_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 ("Wang_3-4_WT-Col-250-microM-KCL-20min-roots_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: Wang_3-4_WT-Col-250-microM-KCL-20min-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolArabidopsis seedlings were grown under hydroponic conditions as previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, approximately 100 seedlings were grown at 25°C with continuous light supported on nylon mesh so that roots were submerged in liquid medium and shoots were above the medium. Seedlings were germinated, then grown in 25 mL of liquid medium containing 2.5 mm (NH4)2 succinate as the nitrogen source, 0.5% (w/v) Suc, and other essential macro- and micronutrients for 9 d, then shifted to 100 mL of fresh medium lacking (NH4)2 succinate for 24 h (note that the initial Cl- concentration was over 2 mm). At day 10, treatments were initiated by adding 250 µL solution of KNO2 or KNO3 (treatment) or KCl (control) to the culture to reach the specified concentrations in the medium. Plants were grown for the specified amount of time and then roots were harvested for RNA extraction.
Sterilisation protocolSeeds were treated with 30% Commercial bleach, 0.05% Triton X-100 for 10 min at room temperature and washed with sterile water 5 times in the hood.
Substrate sterilisation protocolIndividual component stock autoclaved separately; FeEDTA filter-sterilized
MediumThe growth medium contained the following components (final concentration): 10 mM potassium phosphate (pH 6.0), 2.5 mM (NH4)2succinate, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNa2EDTA, 0.5% (w/v) Suc, and micro-nutrients (50 µM H3BO3, 12 µM MnSO4, 1 µM ZnCl2, 1 µM CuSO4, and 0.2 µM Na2MoO4). Component solutions were autoclaved (FeNa2EDTA was filter sterilized) separately and added to sterile deionized water before use.
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Control
in vivo Treatment:
Treatment250 µM KCl
Additional Organism Information:
Sample descriptionWT Col., 250 microM KCL, 20min, roots, replicate 2
Other Information:
ReferencePlant Physiology 145:1735-1745 (2007)

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:0.287051
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:
RawQ2.12
NoiseAvg:2.86,Stdev:0.10,Max:3.3,Min:2.7
BackgroundAvg:61.00,Stdev:0.38,Max:62.1,Min:60.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.287051
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_3-5_WT-Col-250-microM-KNO3-20min-roots_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 ("Wang_3-5_WT-Col-250-microM-KNO3-20min-roots_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: Wang_3-5_WT-Col-250-microM-KNO3-20min-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolArabidopsis seedlings were grown under hydroponic conditions as previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, approximately 100 seedlings were grown at 25°C with continuous light supported on nylon mesh so that roots were submerged in liquid medium and shoots were above the medium. Seedlings were germinated, then grown in 25 mL of liquid medium containing 2.5 mm (NH4)2 succinate as the nitrogen source, 0.5% (w/v) Suc, and other essential macro- and micronutrients for 9 d, then shifted to 100 mL of fresh medium lacking (NH4)2 succinate for 24 h (note that the initial Cl- concentration was over 2 mm). At day 10, treatments were initiated by adding 250 µL solution of KNO2 or KNO3 (treatment) or KCl (control) to the culture to reach the specified concentrations in the medium. Plants were grown for the specified amount of time and then roots were harvested for RNA extraction.
Sterilisation protocolSeeds were treated with 30% Commercial bleach, 0.05% Triton X-100 for 10 min at room temperature and washed with sterile water 5 times in the hood.
Substrate sterilisation protocolIndividual component stock autoclaved separately; FeEDTA filter-sterilized
MediumThe growth medium contained the following components (final concentration): 10 mM potassium phosphate (pH 6.0), 2.5 mM (NH4)2succinate, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNa2EDTA, 0.5% (w/v) Suc, and micro-nutrients (50 µM H3BO3, 12 µM MnSO4, 1 µM ZnCl2, 1 µM CuSO4, and 0.2 µM Na2MoO4). Component solutions were autoclaved (FeNa2EDTA was filter sterilized) separately and added to sterile deionized water before use.
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment250 µM KNO3
Additional Organism Information:
Sample descriptionWT Col., 250 microM KNO3, 20min, roots, replicate 2
Other Information:
ReferencePlant Physiology 145:1735-1745 (2007)

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:0.374929
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:
RawQ2.54
NoiseAvg:3.01,Stdev:0.10,Max:3.3,Min:2.8
BackgroundAvg:69.70,Stdev:0.61,Max:71.8,Min:67.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.374929
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_3-2_WT-Col-250-microM-KNO3-20min-roots_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 ("Wang_3-2_WT-Col-250-microM-KNO3-20min-roots_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: Wang_3-2_WT-Col-250-microM-KNO3-20min-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolArabidopsis seedlings were grown under hydroponic conditions as previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, approximately 100 seedlings were grown at 25°C with continuous light supported on nylon mesh so that roots were submerged in liquid medium and shoots were above the medium. Seedlings were germinated, then grown in 25 mL of liquid medium containing 2.5 mm (NH4)2 succinate as the nitrogen source, 0.5% (w/v) Suc, and other essential macro- and micronutrients for 9 d, then shifted to 100 mL of fresh medium lacking (NH4)2 succinate for 24 h (note that the initial Cl- concentration was over 2 mm). At day 10, treatments were initiated by adding 250 µL solution of KNO2 or KNO3 (treatment) or KCl (control) to the culture to reach the specified concentrations in the medium. Plants were grown for the specified amount of time and then roots were harvested for RNA extraction.
Sterilisation protocolSeeds were treated with 30% Commercial bleach, 0.05% Triton X-100 for 10 min at room temperature and washed with sterile water 5 times in the hood.
Substrate sterilisation protocolIndividual component stock autoclaved separately; FeEDTA filter-sterilized
MediumThe growth medium contained the following components (final concentration): 10 mM potassium phosphate (pH 6.0), 2.5 mM (NH4)2succinate, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNa2EDTA, 0.5% (w/v) Suc, and micro-nutrients (50 µM H3BO3, 12 µM MnSO4, 1 µM ZnCl2, 1 µM CuSO4, and 0.2 µM Na2MoO4). Component solutions were autoclaved (FeNa2EDTA was filter sterilized) separately and added to sterile deionized water before use.
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment250 µM KNO3
Additional Organism Information:
Sample descriptionWT Col., 250 microM KNO3, 20min, roots, replicate 1
Other Information:
ReferencePlant Physiology 145:1735-1745 (2007)

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:0.314943
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:
RawQ2.59
NoiseAvg:3.35,Stdev:0.11,Max:3.8,Min:3.1
BackgroundAvg:72.26,Stdev:0.41,Max:73.4,Min:71.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.314943
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_3-3_WT-Col-250-microM-KNO2-20min-roots_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 ("Wang_3-3_WT-Col-250-microM-KNO2-20min-roots_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: Wang_3-3_WT-Col-250-microM-KNO2-20min-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolArabidopsis seedlings were grown under hydroponic conditions as previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, approximately 100 seedlings were grown at 25°C with continuous light supported on nylon mesh so that roots were submerged in liquid medium and shoots were above the medium. Seedlings were germinated, then grown in 25 mL of liquid medium containing 2.5 mm (NH4)2 succinate as the nitrogen source, 0.5% (w/v) Suc, and other essential macro- and micronutrients for 9 d, then shifted to 100 mL of fresh medium lacking (NH4)2 succinate for 24 h (note that the initial Cl- concentration was over 2 mm). At day 10, treatments were initiated by adding 250 µL solution of KNO2 or KNO3 (treatment) or KCl (control) to the culture to reach the specified concentrations in the medium. Plants were grown for the specified amount of time and then roots were harvested for RNA extraction.
Sterilisation protocolSeeds were treated with 30% Commercial bleach, 0.05% Triton X-100 for 10 min at room temperature and washed with sterile water 5 times in the hood.
Substrate sterilisation protocolIndividual component stock autoclaved separately; FeEDTA filter-sterilized
MediumThe growth medium contained the following components (final concentration): 10 mM potassium phosphate (pH 6.0), 2.5 mM (NH4)2succinate, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNa2EDTA, 0.5% (w/v) Suc, and micro-nutrients (50 µM H3BO3, 12 µM MnSO4, 1 µM ZnCl2, 1 µM CuSO4, and 0.2 µM Na2MoO4). Component solutions were autoclaved (FeNa2EDTA was filter sterilized) separately and added to sterile deionized water before use.
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment250 µM KNO2
Additional Organism Information:
Sample descriptionWT Col., 250 microM KNO2, 20min, roots, replicate 1
Other Information:
ReferencePlant Physiology 145:1735-1745 (2007)

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:0.421418
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:
RawQ2.70
NoiseAvg:3.41,Stdev:0.11,Max:3.7,Min:3.1
BackgroundAvg:76.49,Stdev:0.90,Max:78.7,Min:74.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.421418
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_3-1_WT-Col-250-microM-KCL-20min-roots_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 ("Wang_3-1_WT-Col-250-microM-KCL-20min-roots_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: Wang_3-1_WT-Col-250-microM-KCL-20min-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolArabidopsis seedlings were grown under hydroponic conditions as previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, approximately 100 seedlings were grown at 25°C with continuous light supported on nylon mesh so that roots were submerged in liquid medium and shoots were above the medium. Seedlings were germinated, then grown in 25 mL of liquid medium containing 2.5 mm (NH4)2 succinate as the nitrogen source, 0.5% (w/v) Suc, and other essential macro- and micronutrients for 9 d, then shifted to 100 mL of fresh medium lacking (NH4)2 succinate for 24 h (note that the initial Cl- concentration was over 2 mm). At day 10, treatments were initiated by adding 250 µL solution of KNO2 or KNO3 (treatment) or KCl (control) to the culture to reach the specified concentrations in the medium. Plants were grown for the specified amount of time and then roots were harvested for RNA extraction.
Sterilisation protocolSeeds were treated with 30% Commercial bleach, 0.05% Triton X-100 for 10 min at room temperature and washed with sterile water 5 times in the hood.
Substrate sterilisation protocolIndividual component stock autoclaved separately; FeEDTA filter-sterilized
MediumThe growth medium contained the following components (final concentration): 10 mM potassium phosphate (pH 6.0), 2.5 mM (NH4)2succinate, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNa2EDTA, 0.5% (w/v) Suc, and micro-nutrients (50 µM H3BO3, 12 µM MnSO4, 1 µM ZnCl2, 1 µM CuSO4, and 0.2 µM Na2MoO4). Component solutions were autoclaved (FeNa2EDTA was filter sterilized) separately and added to sterile deionized water before use.
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Control
in vivo Treatment:
Treatment250 µM KCl
Additional Organism Information:
Sample descriptionWT Col., 250 microM KCL, 20min, roots, replicate 1
Other Information:
ReferencePlant Physiology 145:1735-1745 (2007)

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:0.222269
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:
RawQ2.41
NoiseAvg:3.24,Stdev:0.18,Max:3.9,Min:2.8
BackgroundAvg:64.39,Stdev:0.98,Max:67.4,Min:62.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.222269
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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