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Experiment: Gibberellin responsive genes in Arabidopsis

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-423

The aim of the experiment was to identify early gibberellin (GA) responsive genes in the roots of an Arabidopsis GA deficient mutant. The GA deficient mutant used in this study is a transgenic line overexpressing the PcGA2ox1 gene. This mutant has an identical phenotype to ga1-3, but it does not require exogenous GA treatment for germination. Seeds were germinated on 1xMS + 1% (w/v) sucrose plates containing 0.7% gelrite, and grown under continous light. The plates were orientated vertically. After six days growth the plates were transferred to acclimatisation baths containing 1 x MS liquid media so that the roots were submerged. After 24 hours acclimatisation plates were treated with or without 2uM GA4 for 0, 30, 60 and 180 minutes. Root tips (up to the end of the elongation zone) were harvested from these plants (approx 150 per sample), giving a total of 7 experimental samples (1: untreated, 2: 30 mins GA4, 3: 30 mins untreated, 4: 60 mins GA4, 5: 60 mins untreated, 6: 180 mins GA4, 7: 180 mins untreated. Three biological replicates were performed giving a total of 21 root samples. Total RNA was isolated from the roots using the QIAGEN RNeasy method.

About the Experimenter

Name:Dr Steve Thomas
Head of Lab Name:Dr Steve Thomas
Lab:
Address:Rothamsted Research
Harpenden
Herts.
Postcode: AL5 2JQ
Country: UK
 
Telephone Number: 01582763133
Fax Number: 01582763010

All of the data available in this website/database is free, and you are free to do whatever you please with it. If you intend to publish work based on any of this data, please acknowledge us, contact the experimenter above, and either acknowledge them or use them as co-authors in the work.

About this Experiment

Experiment Type: compound_treatment_design;time_series_design;
Number of Slides:21
 
Experimental Parameters:
parametercompound_based_treatment
parameter timepoint
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Thomas: Gibberellin responsive genes in Arabidopsis_genome

Slide: Thomas_1-21_GA4-180mins_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 ("Thomas_1-21_GA4-180mins_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: Thomas_1-21_GA4-180mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: 1I_GA4_3h_3
Stock Code:
Genetic Background: Col-0
Age: 180 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:2.395636558533
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:
RawQ1.09
NoiseAvg:1.12,Stdev:0.05,Max:1.2,Min:1.0
Central-Avg:3943,Count:9
Corner+Avg:71,Count:32
Corner-Avg:5390,Count:32
BackgroundAvg:32.04,Stdev:0.39,Max:32.9,Min:31.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.395636558533
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-20_GA4-60mins_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 ("Thomas_1-20_GA4-60mins_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: Thomas_1-20_GA4-60mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: 1I_GA4_60min_3
Stock Code:
Genetic Background: Col-0
Age: 60 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.09562253952
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:2.09,Stdev:0.07,Max:2.3,Min:1.8
Central-Avg:5741,Count:9
Corner+Avg:54,Count:32
Corner-Avg:5777,Count:32
BackgroundAvg:49.32,Stdev:0.32,Max:50.0,Min:48.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.095622539520
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-19_GA4-30mins_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 ("Thomas_1-19_GA4-30mins_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: Thomas_1-19_GA4-30mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: 1I_GA4_30min_3
Stock Code:
Genetic Background: Col-0
Age: 30 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.368994832039
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:
RawQ1.36
NoiseAvg:1.55,Stdev:0.05,Max:1.7,Min:1.5
Central-Avg:5828,Count:9
Corner+Avg:98,Count:32
Corner-Avg:7315,Count:32
BackgroundAvg:39.55,Stdev:0.37,Max:40.5,Min:38.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.368994832039
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-18_Control-180mins_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 ("Thomas_1-18_Control-180mins_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: Thomas_1-18_Control-180mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: 1I_con_3h_3
Stock Code:
Genetic Background: Col-0
Age: 180 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.391520023346
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:
RawQ1.73
NoiseAvg:1.91,Stdev:0.07,Max:2.2,Min:1.8
Central-Avg:7323,Count:9
Corner+Avg:75,Count:32
Corner-Avg:8811,Count:32
BackgroundAvg:48.41,Stdev:0.26,Max:49.1,Min:47.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.391520023346
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-17_Control-60mins_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 ("Thomas_1-17_Control-60mins_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: Thomas_1-17_Control-60mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: 1I_con_60min_3
Stock Code:
Genetic Background: Col-0
Age: 60 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.623662233353
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.52
NoiseAvg:1.61,Stdev:0.05,Max:1.8,Min:1.5
Central-Avg:5677,Count:9
Corner+Avg:57,Count:32
Corner-Avg:6755,Count:32
BackgroundAvg:42.27,Stdev:0.44,Max:43.3,Min:41.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.623662233353
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-16_Control-30mins_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 ("Thomas_1-16_Control-30mins_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: Thomas_1-16_Control-30mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 30 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:2.797477722168
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:
RawQ1.59
NoiseAvg:1.63,Stdev:0.03,Max:1.7,Min:1.5
Central-Avg:6824,Count:9
Corner+Avg:73,Count:32
Corner-Avg:8063,Count:32
BackgroundAvg:43.54,Stdev:0.48,Max:44.7,Min:42.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.797477722168
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-15_Untreated-control_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 ("Thomas_1-15_Untreated-control_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: Thomas_1-15_Untreated-control_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: zero timepoint
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:3.620378732681
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:
RawQ1.31
NoiseAvg:1.28,Stdev:0.03,Max:1.4,Min:1.2
Central-Avg:6629,Count:9
Corner+Avg:62,Count:32
Corner-Avg:7894,Count:32
BackgroundAvg:36.59,Stdev:0.20,Max:37.2,Min:36.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF3.620378732681
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-1_Untreated-control_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 ("Thomas_1-1_Untreated-control_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: Thomas_1-1_Untreated-control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: zero timepoint
Growth Conditions:
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (200µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: 7 days after sowing the seeds and transferring to the growth room

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:3.234475374222
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:
RawQ1.30
NoiseAvg:1.25,Stdev:0.05,Max:1.3,Min:1.1
Central-Avg:5000,Count:9
Corner+Avg:56,Count:32
Corner-Avg:6413,Count:32
BackgroundAvg:35.52,Stdev:0.12,Max:35.8,Min:35.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF3.234475374222
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-2_Control-30mins_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 ("Thomas_1-2_Control-30mins_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: Thomas_1-2_Control-30mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 30 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: the start time was when the gibberellin hormone or water control was applied to the plants

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.818225622177
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:
RawQ1.78
NoiseAvg:2.09,Stdev:0.05,Max:2.4,Min:2.0
Central-Avg:6008,Count:9
Corner+Avg:70,Count:32
Corner-Avg:7535,Count:32
BackgroundAvg:49.41,Stdev:0.72,Max:50.7,Min:47.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.818225622177
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-3_Control-60mins_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 ("Thomas_1-3_Control-60mins_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: Thomas_1-3_Control-60mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 60 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: the start time was when the gibberellin hormone or water control was applied to the plants

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.604666471481
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:
RawQ1.61
NoiseAvg:1.88,Stdev:0.43,Max:4.6,Min:1.5
Central-Avg:6122,Count:9
Corner+Avg:71,Count:32
Corner-Avg:8379,Count:32
BackgroundAvg:43.87,Stdev:0.45,Max:45.3,Min:42.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.604666471481
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-4_Control-180mins_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 ("Thomas_1-4_Control-180mins_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: Thomas_1-4_Control-180mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 180 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: the start time was when the gibberellin hormone or water control was applied to the plants

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.757863998413
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:
RawQ1.49
NoiseAvg:1.63,Stdev:0.13,Max:2.3,Min:1.4
Central-Avg:4993,Count:9
Corner+Avg:61,Count:32
Corner-Avg:6791,Count:32
BackgroundAvg:42.27,Stdev:0.35,Max:43.3,Min:41.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.757863998413
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-5_GA4-30mins_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 ("Thomas_1-5_GA4-30mins_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: Thomas_1-5_GA4-30mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 30 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: the start time was when the gibberellin hormone or water control was applied to the plants

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.730687379837
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:
RawQ1.76
NoiseAvg:1.95,Stdev:0.07,Max:2.2,Min:1.8
Central-Avg:4912,Count:9
Corner+Avg:51,Count:32
Corner-Avg:5800,Count:32
BackgroundAvg:48.99,Stdev:0.46,Max:50.1,Min:47.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.730687379837
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-6_GA4-60mins_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 ("Thomas_1-6_GA4-60mins_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: Thomas_1-6_GA4-60mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 60 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: the start time was when the gibberellin hormone or water control was applied to the plants

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.245880842209
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:
RawQ1.69
NoiseAvg:1.72,Stdev:0.07,Max:2.1,Min:1.6
Central-Avg:5498,Count:9
Corner+Avg:62,Count:32
Corner-Avg:7419,Count:32
BackgroundAvg:47.50,Stdev:0.25,Max:47.9,Min:46.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.245880842209
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-7_GA4-180mins_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 ("Thomas_1-7_GA4-180mins_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: Thomas_1-7_GA4-180mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 180 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
Other Information: the start time was when the gibberellin hormone or water control was applied to the plants

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:2.441752433777
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:
RawQ1.28
NoiseAvg:1.29,Stdev:0.04,Max:1.4,Min:1.2
Central-Avg:6324,Count:9
Corner+Avg:61,Count:32
Corner-Avg:7887,Count:32
BackgroundAvg:36.58,Stdev:0.12,Max:36.8,Min:36.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.441752433777
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-8_Untreated-control_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 ("Thomas_1-8_Untreated-control_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: Thomas_1-8_Untreated-control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: zero timepoint
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.018849253654
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:
RawQ1.80
NoiseAvg:2.13,Stdev:0.15,Max:3.1,Min:2.0
Central-Avg:4789,Count:9
Corner+Avg:68,Count:32
Corner-Avg:6598,Count:32
BackgroundAvg:50.01,Stdev:0.18,Max:50.5,Min:49.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.018849253654
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-9_Control-30mins_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 ("Thomas_1-9_Control-30mins_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: Thomas_1-9_Control-30mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 30 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.244500517845
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:
RawQ1.65
NoiseAvg:1.81,Stdev:0.05,Max:2.0,Min:1.7
Central-Avg:6297,Count:9
Corner+Avg:66,Count:32
Corner-Avg:7264,Count:32
BackgroundAvg:46.81,Stdev:0.64,Max:47.8,Min:44.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.244500517845
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-10_Control-60mins_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 ("Thomas_1-10_Control-60mins_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: Thomas_1-10_Control-60mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 60 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.771491289139
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:
RawQ1.41
NoiseAvg:1.69,Stdev:0.26,Max:3.0,Min:1.4
Central-Avg:4481,Count:9
Corner+Avg:53,Count:32
Corner-Avg:6060,Count:32
BackgroundAvg:39.57,Stdev:0.36,Max:40.3,Min:38.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.771491289139
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-11_Control-180mins_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Thomas_1-11_Control-180mins_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: Thomas_1-11_Control-180mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 180 minute (water treated control)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.647121787071
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.55
NoiseAvg:1.65,Stdev:0.03,Max:1.8,Min:1.6
Central-Avg:5073,Count:9
Corner+Avg:58,Count:32
Corner-Avg:6618,Count:32
BackgroundAvg:44.92,Stdev:0.23,Max:45.6,Min:44.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.647121787071
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-12_GA4-30mins_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 ("Thomas_1-12_GA4-30mins_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: Thomas_1-12_GA4-30mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 30 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:1.860856175423
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:
RawQ1.33
NoiseAvg:1.41,Stdev:0.05,Max:1.6,Min:1.3
Central-Avg:5544,Count:9
Corner+Avg:60,Count:32
Corner-Avg:7122,Count:32
BackgroundAvg:37.77,Stdev:0.29,Max:38.4,Min:36.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.860856175423
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-13_GA4-60mins_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 ("Thomas_1-13_GA4-60mins_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: Thomas_1-13_GA4-60mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 60 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:0.761094689369
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:2.17,Stdev:0.06,Max:2.3,Min:2.0
Central-Avg:5349,Count:9
Corner+Avg:71,Count:32
Corner-Avg:6186,Count:32
BackgroundAvg:50.64,Stdev:0.27,Max:51.3,Min:49.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.761094689369
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Thomas_1-14_GA4-180mins_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 ("Thomas_1-14_GA4-180mins_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: Thomas_1-14_GA4-180mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: GA2oxOE
Stock Code:
Genetic Background: Col-0
Age: 180 minute (gibberellin treated sample)
Growth Conditions:
Growth medium1x Murashige and Skoog basal salt mixture
Average humidity60%
Average temperature22oC
LocationGrowth Room
Growth substrateGelrite
Average temperature22oC
Average humidity60%
StratificationAfter sterilisation, the seeds were incubated in water at 4C for 72 hours, in the dark.
SterilisationAll seeds were placed in a 1.5ml eppendorf tube containing 70% ethanol with shaking for 3 minutes. The ethanol was removed and replaced with 10% bleach/0.1% Tween-20 for a further 3 minutes . The samples were washed by rinsing with sterile H2O for 2 minutes; this was repeated 4 times.
Growth protocolGA2oxOE seeds were plated onto plates containing 0.7% Gelrite (Duchefa)0.05% MES (Sigma), 1x Murashige and Skoog (Duchefa), 1% Sucrose (Duchefa) at pH 5.8. The plates were stood at an angle of 70º from the horizontal. The plants were grown under continuous light (150µmol-2.sec-1)at 22C. After six days, the lids of the plates were removed and the plates transferred to a treatment bath containing 0.05% MES, 1x Murashige and Skoog, allowing only the roots to be submerged. The plants were grown for a further 24 hours under the same growth conditions. The plates were removed from the 1xMS/0.05% MES solution and the root tips, including the elongation zone, harvested using a razor blade and stored in liquid nitrogen. Approximately 150 root tips were isolated. The root tips were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Developmental Stage:
Developmental stage(Source: Boyes key(Paradigm Genetics))1.03
Genetic Variation: T-DNA gene knock out
Tissue: root tips
in vivo Treatment:
Treatment2uM GA4
Additional Organism Information:
Sample descriptionPlants were 7 day-old Arabidopsis seedlings grown on MS plates. The root tips were used to prepare the total RNA sample. The tip included the elongation zone, but not the differentiation zone.
allelePcGA2ox1 (EMBL accession number: At132438)
Separation Technique: root tip was removed by cutting it off with a razor blade
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:

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:0.890478909016
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:1.96,Stdev:0.04,Max:2.1,Min:1.8
Central-Avg:5089,Count:9
Corner+Avg:65,Count:32
Corner-Avg:6247,Count:32
BackgroundAvg:46.53,Stdev:0.33,Max:47.1,Min:45.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.890478909016
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team