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Experiment: Differential gene expression patterns in the phosphate deficient mutant, pho 1

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-102

Background:

The UK horticultural and agricultural industries routinely apply large amounts of inorganic fertiliser to maintain crop yield and quality, since chemical assays of soil nutrients are unreliable. Excessive fertiliser applications are costly and can lead to unnecessary pollution. A possible solution is to use sensor (GM or non-GM) technologies that exploit the changes in plant gene expression under incipient nutrient deficiency.

Aim:

The aim of this project is to use mutants with reduced leaf phosphate contents to identify genes upregulated in response to phosphate stress. Preliminary gene expression analysis has identified several phosphate responsive genes to be upregulated in the pho1 mutant. However, further replicates of the experiment are required to confirm these changes.

Methods:

Arabidopsis mutant pho1 (N8507) and its parent ecotype Columbia 2 (N907) will be grown on MS agar under identical conditions. RNA will be extracted from the rosette leaves of both parent and mutant and the same growth stage. By comparing the expression profiles, we will be able to differentiate between genes that are upregulated in leaves experiencing phosphate stress. Previously, two GeneChips have been used (mutant and parent) to provide preliminary data. A further two biological replicates are now required to confirm these results. Promoters and transcripts of these genes will underpin the development of novel sensor technologies, and knowledge of the gene expression profiles will improve our understanding of the physiology of plant mineral nutrition.

About the Experimenter

Name:Mr John Hammond
Head of Lab Name:Prof Malcolm Bennett
Lab:
Institute: University of Nottingham
Address:Plant Sciences Division
School of Biosciences
University of Nottingham
Sutton Bonington Campus
Loughborough
Postcode: LE12 5RD
Country: UK
 
Telephone Number: 01789 470382
Fax Number: 01789 470552

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: genetic_modification_design
Number of Slides:6
 
Experimental Parameters:
parametergene_knock_out
Quality Control Measures Taken:
no-plants-pooled20
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Hammond_genome_Exp2_RepSet1

Slide: Hammond_2-1_Col2wildtype_Rep1_ATH1

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

Summary Information

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

  • Extract 1 ("Hammond_2-1_Col2wildtype_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: Hammond_2-1_Col2wildtype_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-2
Stock Code: N907
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity80 %
Temperature24 degrees C
Lighting(Source: 50-80 µmol m-2 s-1)16 h photoperiod
Medium0.8 % agar containing MS salts and 1 % sucrose
NutrientsMS salts and 1 % sucrose
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: Rosette leaves
Plant Ontology terms for this tissue type: 
Unknown PO Term!
Diseased: Normal
Additional Organism Information:
growth_conditionsSeeds were first washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50 % (v/v) domestic bleach/water. Seeds were then rinsed three times in sterile distilled water and imbibed for 3-5 days at 4 degrees C to break dormancy. Seeds were grown in un-vented, polycarbonate culture boxes on 75 ml of 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (Murashige and Skoog, 1962). Illumination was provided by a bank of 100W 84 cool fluorescent tubes (Philips, Eindhoven, Netherlands) giving an photon flux density between 400 and 700 nm of 50-80 µmol photons m-2 s-1 at plant height. Once plant reached the desired growth stage, rosette leaves were snap frozen in liquid nitrogen and total RNA extracted.
Other Information:
experimental_backgroundMutant: Pho1 (N8507). The identification of this mutant has previously been described by Poirier et al., (1991) Plant Physiol., 97, 1087-1093. The identification of the pho1 gene has recently been described by Hamburger et al., (2002) Plant Cell, 14, 889-902.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2003-08-08

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:0.499358
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:
RawQ3.08
NoiseAvg:4.45,Stdev:0.38,Max:5.7,Min:3.7
BackgroundAvg:82.64,Stdev:3.84,Max:94.8,Min:76.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.499358
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hammond_2-2_pho1mutant_Rep1_ATH1

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

Summary Information

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

  • Extract 1 ("Hammond_2-2_pho1mutant_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: Hammond_2-2_pho1mutant_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pho1
Stock Code: N8507
Genetic Background: Col-2
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity80 %
Temperature24 degrees C
Lighting(Source: 50-80 µmol m-2 s-1)16 h photoperiod
Medium0.8 % agar containing MS salts and 1 % sucrose
NutrientsMS salts and 1 % sucrose
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Genetic Variation: EMS mutant
Tissue: Rosette leaves
Plant Ontology terms for this tissue type: 
Unknown PO Term!
Diseased: Normal
Additional Organism Information:
growth_conditionsSeeds were first washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50 % (v/v) domestic bleach/water. Seeds were then rinsed three times in sterile distilled water and imbibed for 3-5 days at 4 degrees C to break dormancy. Seeds were grown in un-vented, polycarbonate culture boxes on 75 ml of 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (Murashige and Skoog, 1962). Illumination was provided by a bank of 100W 84 cool fluorescent tubes (Philips, Eindhoven, Netherlands) giving an photon flux density between 400 and 700 nm of 50-80 µmol photons m-2 s-1 at plant height. Once plant reached the desired growth stage, rosette leaves were snap frozen in liquid nitrogen and total RNA extracted.
Other Information:
experimental_backgroundMutant: Pho1 (N8507). The identification of this mutant has previously been described by Poirier et al., (1991) Plant Physiol., 97, 1087-1093. The identification of the pho1 gene has recently been described by Hamburger et al., (2002) Plant Cell, 14, 889-902.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2003-08-08

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:0.561643
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.96
NoiseAvg:4.00,Stdev:0.23,Max:4.6,Min:3.5
BackgroundAvg:79.74,Stdev:4.03,Max:90.5,Min:71.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.561643
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Hybridisation Set: Hammond_genome_Exp2_RepSet2

Slide: Hammond_2-3_Col2wildtype_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Hammond_2-3_Col2wildtype_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: Hammond_2-3_Col2wildtype_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-2
Stock Code: N907
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity80 %
Temperature24 degrees C
Lighting(Source: 50-80 µmol m-2 s-1)16 h photoperiod
Medium0.8 % agar containing MS salts and 1 % sucrose
NutrientsMS salts and 1 % sucrose
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: Rosette leaves
Plant Ontology terms for this tissue type: 
Unknown PO Term!
Diseased: Normal
Additional Organism Information:
growth_conditionsSeeds were first washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50 % (v/v) domestic bleach/water. Seeds were then rinsed three times in sterile distilled water and imbibed for 3-5 days at 4 degrees C to break dormancy. Seeds were grown in un-vented, polycarbonate culture boxes on 75 ml of 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (Murashige and Skoog, 1962). Illumination was provided by a bank of 100W 84 cool fluorescent tubes (Philips, Eindhoven, Netherlands) giving an photon flux density between 400 and 700 nm of 50-80 µmol photons m-2 s-1 at plant height. Once plant reached the desired growth stage, rosette leaves were snap frozen in liquid nitrogen and total RNA extracted.
Other Information:
experimental_backgroundMutant: Pho1 (N8507). The identification of this mutant has previously been described by Poirier et al., (1991) Plant Physiol., 97, 1087-1093. The identification of the pho1 gene has recently been described by Hamburger et al., (2002) Plant Cell, 14, 889-902.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2003-12-11

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:0.385108
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.35
NoiseAvg:3.56,Stdev:0.17,Max:4.0,Min:3.2
BackgroundAvg:61.84,Stdev:1.57,Max:65.6,Min:57.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.385108
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hammond_2-4_pho1mutant_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Hammond_2-4_pho1mutant_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: Hammond_2-4_pho1mutant_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pho1
Stock Code: N8507
Genetic Background: Col-2
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity80 %
Temperature24 degrees C
Lighting(Source: 50-80 µmol m-2 s-1)16 h photoperiod
Medium0.8 % agar containing MS salts and 1 % sucrose
NutrientsMS salts and 1 % sucrose
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Genetic Variation: EMS mutant
Tissue: Rosette leaves
Plant Ontology terms for this tissue type: 
Unknown PO Term!
Diseased: Normal
Additional Organism Information:
growth_conditionsSeeds were first washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50 % (v/v) domestic bleach/water. Seeds were then rinsed three times in sterile distilled water and imbibed for 3-5 days at 4 degrees C to break dormancy. Seeds were grown in un-vented, polycarbonate culture boxes on 75 ml of 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (Murashige and Skoog, 1962). Illumination was provided by a bank of 100W 84 cool fluorescent tubes (Philips, Eindhoven, Netherlands) giving an photon flux density between 400 and 700 nm of 50-80 µmol photons m-2 s-1 at plant height. Once plant reached the desired growth stage, rosette leaves were snap frozen in liquid nitrogen and total RNA extracted.
Other Information:
experimental_backgroundMutant: Pho1 (N8507). The identification of this mutant has previously been described by Poirier et al., (1991) Plant Physiol., 97, 1087-1093. The identification of the pho1 gene has recently been described by Hamburger et al., (2002) Plant Cell, 14, 889-902.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2003-12-11

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:0.495779
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.39
NoiseAvg:3.57,Stdev:0.28,Max:4.6,Min:3.0
BackgroundAvg:58.96,Stdev:1.78,Max:65.1,Min:55.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.495779
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Hybridisation Set: Hammond_genome_Exp2_RepSet3

Slide: Hammond_2-5_Col2wildtype_Rep3_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 ("Hammond_2-5_Col2wildtype_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Hammond_2-5_Col2wildtype_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-2
Stock Code: N907
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity80 %
Temperature24 degrees C
Lighting(Source: 50-80 µmol m-2 s-1)16 h photoperiod
Medium0.8 % agar containing MS salts and 1 % sucrose
NutrientsMS salts and 1 % sucrose
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: Rosette leaves
Plant Ontology terms for this tissue type: 
Unknown PO Term!
Diseased: Normal
Additional Organism Information:
growth_conditionsSeeds were first washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50 % (v/v) domestic bleach/water. Seeds were then rinsed three times in sterile distilled water and imbibed for 3-5 days at 4 degrees C to break dormancy. Seeds were grown in un-vented, polycarbonate culture boxes on 75 ml of 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (Murashige and Skoog, 1962). Illumination was provided by a bank of 100W 84 cool fluorescent tubes (Philips, Eindhoven, Netherlands) giving an photon flux density between 400 and 700 nm of 50-80 µmol photons m-2 s-1 at plant height. Once plant reached the desired growth stage, rosette leaves were snap frozen in liquid nitrogen and total RNA extracted.
Other Information:
experimental_backgroundMutant: Pho1 (N8507). The identification of this mutant has previously been described by Poirier et al., (1991) Plant Physiol., 97, 1087-1093. The identification of the pho1 gene has recently been described by Hamburger et al., (2002) Plant Cell, 14, 889-902.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2003-12-11

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:0.358104
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.54
NoiseAvg:4.16,Stdev:0.16,Max:4.7,Min:3.8
BackgroundAvg:66.32,Stdev:1.47,Max:70.0,Min:62.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.358104
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hammond_2-6_pho1mutant_Rep3_ATH1

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

Summary Information

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

  • Extract 1 ("Hammond_2-6_pho1mutant_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Hammond_2-6_pho1mutant_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pho1
Stock Code: N8507
Genetic Background: Col-2
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidity80 %
Temperature24 degrees C
Lighting(Source: 50-80 µmol m-2 s-1)16 h photoperiod
Medium0.8 % agar containing MS salts and 1 % sucrose
NutrientsMS salts and 1 % sucrose
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Genetic Variation: EMS mutant
Tissue: Rosette leaves
Plant Ontology terms for this tissue type: 
Unknown PO Term!
Diseased: Normal
Additional Organism Information:
growth_conditionsSeeds were first washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50 % (v/v) domestic bleach/water. Seeds were then rinsed three times in sterile distilled water and imbibed for 3-5 days at 4 degrees C to break dormancy. Seeds were grown in un-vented, polycarbonate culture boxes on 75 ml of 0.8 % (w/v) agar containing 1 % (w/v) sucrose and a basal salt mix (Murashige and Skoog, 1962). Illumination was provided by a bank of 100W 84 cool fluorescent tubes (Philips, Eindhoven, Netherlands) giving an photon flux density between 400 and 700 nm of 50-80 µmol photons m-2 s-1 at plant height. Once plant reached the desired growth stage, rosette leaves were snap frozen in liquid nitrogen and total RNA extracted.
Other Information:
experimental_backgroundMutant: Pho1 (N8507). The identification of this mutant has previously been described by Poirier et al., (1991) Plant Physiol., 97, 1087-1093. The identification of the pho1 gene has recently been described by Hamburger et al., (2002) Plant Cell, 14, 889-902.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

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

Slide Information

Date Hybridised: 2003-12-11

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:0.445207
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.62
NoiseAvg:4.08,Stdev:0.13,Max:4.6,Min:3.8
BackgroundAvg:70.84,Stdev:2.23,Max:74.9,Min:66.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.445207
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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