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Experiment: Identification of early gibberellin (GA) responsive genes in Arabidopsis hypocotyls

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-482

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 treated with or without 5uM GA4 for 0, 30, 60 and 180 minutes.Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen 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 Anne Gronlund
Head of Lab Name:Dr Steve Thomas
Lab:
Address:West Common
Postcode: AL5 2JQ
Country: United Kingdom
 

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

About this Experiment

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

Slides in this Experiment

Hybridisation Set: Gronlund: Identification of early gibberellin (GA) responsive genes in Arabidopsis hypocotyls_genome

Slide: Gronlund_1-21_BR3+GA-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 ("Gronlund_1-21_BR3+GA-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: Gronlund_1-21_BR3+GA-180mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.995309710503
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.05
NoiseAvg:1.92,Stdev:0.06,Max:2.1,Min:1.7
Central-Avg:4708,Count:9
Corner+Avg:59,Count:32
Corner-Avg:6210,Count:32
BackgroundAvg:48.28,Stdev:0.38,Max:49.1,Min:47.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.995309710503
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-20_BR3-180mins_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 ("Gronlund_1-20_BR3-180mins_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: Gronlund_1-20_BR3-180mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.735281169415
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.12
NoiseAvg:2.17,Stdev:0.08,Max:2.4,Min:2.0
Central-Avg:6520,Count:9
Corner+Avg:71,Count:32
Corner-Avg:7731,Count:32
BackgroundAvg:51.37,Stdev:0.51,Max:52.8,Min:49.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.735281169415
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-19_BR3+GA-60mins_Rep3_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Gronlund_1-19_BR3+GA-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: Gronlund_1-19_BR3+GA-60mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:1.076570153236
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:
RawQ1.97
NoiseAvg:2.01,Stdev:0.04,Max:2.2,Min:1.8
Central-Avg:6903,Count:9
Corner+Avg:64,Count:32
Corner-Avg:7684,Count:32
BackgroundAvg:48.71,Stdev:0.64,Max:50.2,Min:46.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.076570153236
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-18_BR3-60mins_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 ("Gronlund_1-18_BR3-60mins_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: Gronlund_1-18_BR3-60mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.876938521862
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.12
NoiseAvg:2.06,Stdev:0.07,Max:2.3,Min:1.9
Central-Avg:6043,Count:9
Corner+Avg:64,Count:32
Corner-Avg:7016,Count:32
BackgroundAvg:50.10,Stdev:0.48,Max:51.2,Min:48.4
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.876938521862
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-17_BR3+GA-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 ("Gronlund_1-17_BR3+GA-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: Gronlund_1-17_BR3+GA-30mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.80005043745
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:
RawQ1.84
NoiseAvg:1.84,Stdev:0.06,Max:2.1,Min:1.7
Central-Avg:6567,Count:9
Corner+Avg:71,Count:32
Corner-Avg:7978,Count:32
BackgroundAvg:44.71,Stdev:0.55,Max:46.2,Min:43.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.800050437450
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-16_BR3-30mins_Rep3_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Gronlund_1-16_BR3-30mins_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: Gronlund_1-16_BR3-30mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:1.034901976585
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:
RawQ1.96
NoiseAvg:1.96,Stdev:0.04,Max:2.1,Min:1.8
Central-Avg:5461,Count:9
Corner+Avg:61,Count:32
Corner-Avg:6721,Count:32
BackgroundAvg:47.15,Stdev:0.67,Max:48.7,Min:45.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.034901976585
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-15_BR3-0mins_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 ("Gronlund_1-15_BR3-0mins_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: Gronlund_1-15_BR3-0mins_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.895971059799
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.03
NoiseAvg:1.95,Stdev:0.04,Max:2.1,Min:1.9
Central-Avg:6037,Count:9
Corner+Avg:65,Count:32
Corner-Avg:7400,Count:32
BackgroundAvg:49.06,Stdev:0.32,Max:49.9,Min:48.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.895971059799
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-14_BR2+GA-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 ("Gronlund_1-14_BR2+GA-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: Gronlund_1-14_BR2+GA-180mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:1.003904938698
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:
RawQ1.87
NoiseAvg:1.77,Stdev:0.05,Max:1.9,Min:1.7
Central-Avg:5055,Count:9
Corner+Avg:57,Count:32
Corner-Avg:6416,Count:32
BackgroundAvg:44.39,Stdev:0.21,Max:45.0,Min:43.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.003904938698
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-13_BR2-180mins_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 ("Gronlund_1-13_BR2-180mins_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: Gronlund_1-13_BR2-180mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.868624687195
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:
RawQ1.97
NoiseAvg:2.00,Stdev:0.07,Max:2.2,Min:1.8
Central-Avg:6014,Count:9
Corner+Avg:69,Count:32
Corner-Avg:7199,Count:32
BackgroundAvg:47.53,Stdev:0.39,Max:48.8,Min:46.6
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.868624687195
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-12_BR2+GA-60mins_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 ("Gronlund_1-12_BR2+GA-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: Gronlund_1-12_BR2+GA-60mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.617349803448
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.16
NoiseAvg:2.29,Stdev:0.08,Max:2.5,Min:2.1
Central-Avg:7431,Count:9
Corner+Avg:80,Count:32
Corner-Avg:8238,Count:32
BackgroundAvg:52.89,Stdev:0.37,Max:53.8,Min:51.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.617349803448
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-11_BR2-60mins_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 ("Gronlund_1-11_BR2-60mins_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: Gronlund_1-11_BR2-60mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.685896039009
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.11
NoiseAvg:2.10,Stdev:0.03,Max:2.2,Min:2.0
Central-Avg:6116,Count:9
Corner+Avg:69,Count:32
Corner-Avg:7371,Count:32
BackgroundAvg:50.00,Stdev:0.27,Max:50.8,Min:49.4
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.685896039009
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-10_BR2+GA-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 ("Gronlund_1-10_BR2+GA-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: Gronlund_1-10_BR2+GA-30mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:1.047080397606
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.09
NoiseAvg:2.08,Stdev:0.09,Max:2.3,Min:1.9
Central-Avg:5430,Count:9
Corner+Avg:59,Count:32
Corner-Avg:6282,Count:32
BackgroundAvg:49.33,Stdev:0.42,Max:50.6,Min:48.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.047080397606
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-9_BR2-30mins_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 ("Gronlund_1-9_BR2-30mins_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: Gronlund_1-9_BR2-30mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.872069299221
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:
RawQ1.99
NoiseAvg:1.87,Stdev:0.05,Max:2.0,Min:1.7
Central-Avg:5134,Count:9
Corner+Avg:63,Count:32
Corner-Avg:6958,Count:32
BackgroundAvg:46.91,Stdev:0.40,Max:48.0,Min:45.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.872069299221
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-8_BR2-0mins_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 ("Gronlund_1-8_BR2-0mins_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: Gronlund_1-8_BR2-0mins_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.738158941269
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.17
NoiseAvg:2.16,Stdev:0.05,Max:2.4,Min:2.0
Central-Avg:6373,Count:9
Corner+Avg:65,Count:32
Corner-Avg:6952,Count:32
BackgroundAvg:54.21,Stdev:0.52,Max:55.6,Min:52.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.738158941269
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-7_BR1+GA-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 ("Gronlund_1-7_BR1+GA-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: Gronlund_1-7_BR1+GA-180mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.670181035995
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.20
NoiseAvg:2.22,Stdev:0.07,Max:2.4,Min:2.1
Central-Avg:7662,Count:9
Corner+Avg:75,Count:32
Corner-Avg:8514,Count:32
BackgroundAvg:51.06,Stdev:0.67,Max:52.5,Min:49.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.670181035995
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-6_BR1-180mins_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 ("Gronlund_1-6_BR1-180mins_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: Gronlund_1-6_BR1-180mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:1.094629645348
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.09
NoiseAvg:2.15,Stdev:0.08,Max:2.5,Min:2.0
Central-Avg:5430,Count:9
Corner+Avg:59,Count:32
Corner-Avg:6296,Count:32
BackgroundAvg:51.90,Stdev:0.64,Max:53.8,Min:50.6
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.094629645348
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-5_BR1+GA-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 ("Gronlund_1-5_BR1+GA-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: Gronlund_1-5_BR1+GA-60mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.663050115108
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.12
NoiseAvg:2.20,Stdev:0.05,Max:2.3,Min:2.0
Central-Avg:7136,Count:9
Corner+Avg:69,Count:32
Corner-Avg:8025,Count:32
BackgroundAvg:50.39,Stdev:0.35,Max:51.2,Min:49.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.663050115108
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-4_BR1-60mins_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 ("Gronlund_1-4_BR1-60mins_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: Gronlund_1-4_BR1-60mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.830522418022
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.11
NoiseAvg:2.24,Stdev:0.05,Max:2.4,Min:2.1
Central-Avg:6543,Count:9
Corner+Avg:72,Count:32
Corner-Avg:7587,Count:32
BackgroundAvg:51.53,Stdev:0.23,Max:51.9,Min:50.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.830522418022
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-3_BR1+GA-30mins_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 ("Gronlund_1-3_BR1+GA-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: Gronlund_1-3_BR1+GA-30mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.848317742348
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.15
NoiseAvg:2.15,Stdev:0.06,Max:2.3,Min:2.0
Central-Avg:5448,Count:9
Corner+Avg:70,Count:32
Corner-Avg:7326,Count:32
BackgroundAvg:51.98,Stdev:0.34,Max:52.6,Min:50.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.848317742348
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-2_BR1-30mins_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 ("Gronlund_1-2_BR1-30mins_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: Gronlund_1-2_BR1-30mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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: 2008-11-13

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:0.584597229958
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.21
NoiseAvg:2.27,Stdev:0.06,Max:2.4,Min:2.1
Central-Avg:6947,Count:9
Corner+Avg:79,Count:32
Corner-Avg:8380,Count:32
BackgroundAvg:51.22,Stdev:0.40,Max:52.6,Min:50.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.584597229958
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Gronlund_1-1_BR1-0mins_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 ("Gronlund_1-1_BR1-0mins_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: Gronlund_1-1_BR1-0mins_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Pcga2ox1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
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.
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 seedlings were either treated with water or 5 µM GA4 for 0, 30, 60 and 180 min. Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen. The hypocotyls were ground using a TissueLyser (Qiagen) and total RNA extracted using a Qiagen RNeasy kit.
Temperature22 °C average, 22 °C day, 22 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture
LightingConstant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: knock out mutation in Pcga2ox1
Tissue: whole plant
in vivo Treatment: GA4 treatment
Additional Organism Information:
Sample Description
Other Information:
Timecourse start procedure

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:1.047344446182
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.03
NoiseAvg:1.98,Stdev:0.04,Max:2.1,Min:1.9
Central-Avg:5084,Count:9
Corner+Avg:73,Count:32
Corner-Avg:6583,Count:32
BackgroundAvg:47.65,Stdev:0.45,Max:48.9,Min:46.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.047344446182
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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