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Experiment: WT vs NR null mutant high nitrate concn treatment

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-480

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mM nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.

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; genetic_modification_design
Number of Slides:16
 
Experimental Parameters:
parametercompound_based_treatment
parametergene_knock_out
Quality Control Measures Taken:
References:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522.
 
Other Information:

Slides in this Experiment

Hybridisation Set: Wang: WT vs NR null mutant high nitrate concn treatment_genome

Slide: Wang_2-16_Wild-type-Col-5-mM-KNO3-2-hr-shoots_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_2-16_Wild-type-Col-5-mM-KNO3-2-hr-shoots_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_2-16_Wild-type-Col-5-mM-KNO3-2-hr-shoots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KNO3, 2 hr, shoots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.574519
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.86
NoiseAvg:3.98,Stdev:0.18,Max:4.8,Min:3.6
BackgroundAvg:85.57,Stdev:0.81,Max:88.0,Min:84.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.574519
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-15_Wild-type-Col-5-mM-KCL-2-hr-shoots_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 ("Wang_2-15_Wild-type-Col-5-mM-KCL-2-hr-shoots_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_2-15_Wild-type-Col-5-mM-KCL-2-hr-shoots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KCL, 2 hr, shoots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.633584
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.64
NoiseAvg:3.79,Stdev:0.13,Max:4.1,Min:3.5
BackgroundAvg:80.84,Stdev:1.09,Max:83.2,Min:78.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.633584
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-13_Wild-type-Col-5-mM-KCL-2-hr-shoots_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_2-13_Wild-type-Col-5-mM-KCL-2-hr-shoots_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_2-13_Wild-type-Col-5-mM-KCL-2-hr-shoots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KCL, 2 hr, shoots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.562376
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:
RawQ4.06
NoiseAvg:4.44,Stdev:0.18,Max:4.9,Min:3.6
BackgroundAvg:94.21,Stdev:3.47,Max:104.3,Min:85.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.562376
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-14_Wild-type-Col-5-mM-KNO3-2-hr-shoots_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_2-14_Wild-type-Col-5-mM-KNO3-2-hr-shoots_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_2-14_Wild-type-Col-5-mM-KNO3-2-hr-shoots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KNO3, 2 hr, shoots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.714243
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.78
NoiseAvg:3.91,Stdev:0.10,Max:4.3,Min:3.7
BackgroundAvg:91.07,Stdev:1.81,Max:94.9,Min:87.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.714243
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-11_Wild-type-Col-5-mM-KCL-2-hr-roots_Rep2_ATH1

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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_2-11_Wild-type-Col-5-mM-KCL-2-hr-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_2-11_Wild-type-Col-5-mM-KCL-2-hr-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KCL, 2 hr, roots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.535392
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.79
NoiseAvg:4.14,Stdev:0.13,Max:4.5,Min:3.8
BackgroundAvg:80.94,Stdev:1.01,Max:83.6,Min:78.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.535392
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-12_Wild-type-Col-5-mM-KNO3-2-hr-roots_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 ("Wang_2-12_Wild-type-Col-5-mM-KNO3-2-hr-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_2-12_Wild-type-Col-5-mM-KNO3-2-hr-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KNO3, 2 hr, roots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.643888
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.74
NoiseAvg:3.98,Stdev:0.11,Max:4.4,Min:3.7
BackgroundAvg:84.79,Stdev:1.06,Max:87.4,Min:82.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.643888
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-10_Wild-type-Col-5-mM-KNO3-2-hr-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_2-10_Wild-type-Col-5-mM-KNO3-2-hr-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_2-10_Wild-type-Col-5-mM-KNO3-2-hr-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KNO3, 2 hr, roots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.341956
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:
RawQ4.56
NoiseAvg:5.20,Stdev:0.14,Max:5.6,Min:4.8
BackgroundAvg:100.70,Stdev:0.46,Max:102.4,Min:100.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.341956
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-9_Wild-type-Col-5-mM-KCL-2-hr-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_2-9_Wild-type-Col-5-mM-KCL-2-hr-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_2-9_Wild-type-Col-5-mM-KCL-2-hr-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col.-0
Stock Code:
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionWild type Col, 5 mM KCL, 2 hr, roots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.345766
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:
RawQ4.57
NoiseAvg:5.22,Stdev:0.13,Max:5.6,Min:4.9
BackgroundAvg:100.81,Stdev:1.25,Max:103.9,Min:97.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.345766
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-8_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-shoots_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 ("Wang_2-8_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-shoots_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_2-8_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-shoots_Rep2_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KNO3, 2 hr, shoots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.950682
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.59
NoiseAvg:3.40,Stdev:0.11,Max:3.8,Min:3.0
BackgroundAvg:74.64,Stdev:0.59,Max:76.0,Min:72.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.950682
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-7_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-shoots_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 ("Wang_2-7_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-shoots_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_2-7_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-shoots_Rep2_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KCL, 2 hr, shoots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.786088
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.95
NoiseAvg:3.85,Stdev:0.12,Max:4.3,Min:3.5
BackgroundAvg:91.50,Stdev:1.03,Max:94.1,Min:88.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.786088
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-6_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-shoots_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_2-6_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-shoots_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_2-6_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-shoots_Rep1_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KNO3, 2 hr, shoots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.715951
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:
RawQ4.30
NoiseAvg:4.31,Stdev:0.14,Max:4.9,Min:4.1
BackgroundAvg:103.95,Stdev:1.06,Max:106.4,Min:100.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.715951
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-5_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-shoots_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_2-5_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-shoots_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_2-5_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-shoots_Rep1_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: shoots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KCL, 2 hr, shoots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.690553
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.88
NoiseAvg:3.98,Stdev:0.18,Max:4.5,Min:3.4
BackgroundAvg:85.14,Stdev:2.16,Max:89.5,Min:79.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.690553
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-1_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-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_2-1_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-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_2-1_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KCL, 2 hr, roots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.745294
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.40
NoiseAvg:3.54,Stdev:0.13,Max:4.0,Min:3.2
BackgroundAvg:74.94,Stdev:0.82,Max:77.0,Min:72.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.745294
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-2_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-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_2-2_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-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_2-2_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-roots_Rep1_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KNO3, 2 hr, roots, replicate 1
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.91092
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.16
NoiseAvg:3.18,Stdev:0.13,Max:3.5,Min:2.9
BackgroundAvg:67.59,Stdev:1.28,Max:69.8,Min:64.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.910920
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-3_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-roots_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 ("Wang_2-3_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-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_2-3_Nitrate-reductase-null-mutant-5-mM-KCL-2-hr-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Control
in vivo Treatment:
Treatment5 mM KCl
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KCL, 2 hr, roots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.423205
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:
RawQ4.46
NoiseAvg:4.93,Stdev:0.17,Max:5.3,Min:4.4
BackgroundAvg:95.26,Stdev:0.55,Max:96.9,Min:93.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.423205
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Wang_2-4_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-roots_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 ("Wang_2-4_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-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_2-4_Nitrate-reductase-null-mutant-5-mM-KNO3-2-hr-roots_Rep2_ATH1
Organism: Arabidopsis thaliana
Stock Code: N2356
Growth Conditions:
Growth protocolFor the microarray experiments, plants were grown under hydroponic conditions previously described (Plant Physiol. 2003 June; 132(2): 556–567.). Briefly, plants were grown supported on liquid media containing 2.5 mm (ammonium)2succinate (equivalent to 5 mm NH4) as the nitrogen source and 0.5% (w/v) Suc for 10 d at 25°C with continuous light. A 1-mL solution of KNO3 (treatment) or KCl (control) was added to the culture to final concentration of 5 mm; plants were grown for another 2 h then harvested for RNA extraction. For growth on agarose plates, seeds were surface sterilized and then individually placed on 50 mL of agarose medium spaced at 5 mm apart in a 100-mm gridded square petri dish. After 24-h cold treatment at 4°C, the plates were incubated in a 25°C growth room under 16 h light. The medium in the plates is the same as described previously for liquid culture (Plant Physiol. 2003 June; 132(2): 556–567.) with 2.5 mm (ammonium)2succinate, 2.5 mm NH4NO3, or 5 mm KNO3 as the sole nitrogen source as indicated. To propagate the NR-null mutant, seedlings are first grown on agarose plates with 2.5 mm (ammonium)2succinate as the sole nitrogen source for 10 d as described above. Seedlings are then transplanted onto 2-inch pots containing Vermiculite (medium coarse; Therm-O-Rock West, Chandler, AZ) and grown in 16 h light at 23°C. Before transplantation, the Vermiculite pots are rinsed twice from top with distilled water and autoclaved. The pots are then washed twice (about 60 mL/wash) from top with vermiculite pot medium composed of 10 mm KH2PO4/K2HPO4 (pH 6.5), 1 mm (ammonium)2succinate, 2 mm MgSO4, 1 mm CaCl2, 0.125 mm NaFeEDTA, 0.5% (w/v) Suc, 0.125 mm H3BO3, 0.03 mm MnSO4, 2.5 µm ZnSO4, 2.5 µm CuSO4, and 0. 5 µm Na2MoO4. After transplantation, pots are covered with transparent plastic for 5 d (without watering), the cover is removed, and the pots are watered twice weekly from top with vermiculite pot medium (about 100 mL/pot).
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
Developmental Stage:
Developmental stage10 days
Tissue: Roots
Diseased: Treatment
in vivo Treatment:
Treatment5 mM KNO3
Additional Organism Information:
Sample descriptionNitrate reductase null mutant, 5 mM KNO3, 2 hr, roots, replicate 2
Other Information:
ReferencePlant Physiol. 2004 September; 136(1): 2512-2522

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:0.386052
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:
RawQ4.99
NoiseAvg:5.67,Stdev:0.27,Max:6.4,Min:4.9
BackgroundAvg:114.40,Stdev:2.37,Max:121.1,Min:108.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.386052
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team