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Experiment: Cesium Toxicity in Arabidopsis

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-384

Caesium (Cs+) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs+ is chemically similar to potassium (K+), and much is known about K+ transport mechanisms, it is not clear through which K+ transport mechanisms Cs+ is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K+ and Cs+ uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K+ deprivation, when it is expressed.

About the Experimenter

Name:Miss Corrina Hampton
Head of Lab Name:Dr Philip White
Lab:
Institute: Horticulture Research International
Address:Horticulture Research International
Wellesbourne
Warwick
Postcode: CV35 9EF
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: strain_or_line_design
Number of Slides:9
 
Experimental Parameters:
parameterstrain_or_line
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Hampton: 2

Slide: Hampton_2-1_Col-0_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 ("Hampton_2-1_Col-0_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: Hampton_2-1_Col-0_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Tissue: Root
Other Information:

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: 2009-02-02

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:1.130831
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:
RawQ2.01
NoiseAvg:2.58,Stdev:0.08,Max:2.8,Min:2.4
BackgroundAvg:54.35,Stdev:0.90,Max:57.1,Min:52.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.130831
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-2_Col-0_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 ("Hampton_2-2_Col-0_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: Hampton_2-2_Col-0_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Tissue: Root
Other Information:

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: 2009-02-02

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:19.063398
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:
RawQ2.21
NoiseAvg:2.26,Stdev:0.10,Max:2.6,Min:2.0
BackgroundAvg:56.19,Stdev:0.74,Max:58.7,Min:55.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF19.063398
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-3_Col-0_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 ("Hampton_2-3_Col-0_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: Hampton_2-3_Col-0_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Tissue: Root
Other Information:

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: 2009-02-02

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:10.097587
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.83
NoiseAvg:1.80,Stdev:0.06,Max:2.0,Min:1.7
BackgroundAvg:48.77,Stdev:0.46,Max:50.3,Min:48.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF10.097587
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-4_cngc1-3_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 ("Hampton_2-4_cngc1-3_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Hampton_2-4_cngc1-3_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: cngc1-3
Stock Code:
Genetic Background: Col-0
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Genetic Variation: T-DNA gene knock out
Tissue: Root
Additional Organism Information:
Alleles knocked outAt5g53130
Other Information:

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: 2009-02-02

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:1.663377
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.56
NoiseAvg:1.73,Stdev:0.07,Max:1.9,Min:1.5
BackgroundAvg:40.03,Stdev:0.36,Max:40.9,Min:39.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.663377
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-5_cngc1-3_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 ("Hampton_2-5_cngc1-3_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: Hampton_2-5_cngc1-3_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: cngc1-3
Stock Code:
Genetic Background: Col-0
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Genetic Variation: T-DNA gene knock out
Tissue: Root
Additional Organism Information:
Alleles knocked outAt5g53130
Other Information:

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: 2009-02-02

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:1.13553
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.62
NoiseAvg:1.94,Stdev:0.05,Max:2.1,Min:1.8
BackgroundAvg:40.27,Stdev:0.36,Max:41.3,Min:39.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.135530
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-6_cngc1-3_Rep3_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Hampton_2-6_cngc1-3_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: Hampton_2-6_cngc1-3_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: cngc1-3
Stock Code:
Genetic Background: Col-0
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Genetic Variation: T-DNA gene knock out
Tissue: Root
Additional Organism Information:
Alleles knocked outAt5g53130
Other Information:

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: 2009-02-02

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:1.12086
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:
RawQ3.14
NoiseAvg:6.67,Stdev:0.26,Max:7.4,Min:5.7
BackgroundAvg:86.42,Stdev:1.29,Max:90.8,Min:84.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.120860
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-7_cngc19_Rep1_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Hampton_2-7_cngc19_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: Hampton_2-7_cngc19_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: cngc19
Stock Code:
Genetic Background: Col-0
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Genetic Variation: T-DNA gene knock out
Tissue: Root
Additional Organism Information:
Alleles knocked outAt3g17690
Other Information:

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: 2009-02-02

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:1.312624
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.66
NoiseAvg:1.91,Stdev:0.09,Max:2.2,Min:1.7
BackgroundAvg:42.40,Stdev:0.36,Max:43.6,Min:41.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.312624
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-8_cngc19_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 ("Hampton_2-8_cngc19_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: Hampton_2-8_cngc19_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: cngc19
Stock Code:
Genetic Background: Col-0
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Genetic Variation: T-DNA gene knock out
Tissue: Root
Additional Organism Information:
Alleles knocked outAt3g17690
Other Information:

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: 2009-02-02

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:1.215882
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.77
NoiseAvg:2.15,Stdev:0.07,Max:2.3,Min:1.9
BackgroundAvg:47.45,Stdev:0.40,Max:48.5,Min:46.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.215882
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Hampton_2-9_cngc19_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 ("Hampton_2-9_cngc19_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: Hampton_2-9_cngc19_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: cngc19
Stock Code:
Genetic Background: Col-0
Growth Conditions:
SterilisationSeeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 °C to break dormancy.
Growth protocolFollowing imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
LocationGrowth Room
Growth substrateHydroponics
Hydroponic constituentsIn the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker.
Average humidity80%
Average temperature22°C
Growth mediumKH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM
Sample descriptionPlants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 °C prior to the extraction of total RNA.
Developmental Stage:
Devlopmental stage(Source: Boyes Key - Paradigm Genetics)3.90
Genetic Variation: T-DNA gene knock out
Tissue: Root
Additional Organism Information:
Alleles knocked outAt3g17690
Other Information:

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: 2009-02-02

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:1.766719
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.90
NoiseAvg:2.27,Stdev:0.06,Max:2.4,Min:2.1
BackgroundAvg:51.68,Stdev:0.44,Max:52.4,Min:50.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.766719
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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