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Experiment: Expression comparison for white light grown Wt vs knockout arabidopsis

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-477

The aim of this experiment is determine regulation of downstream factors through comparison of Wildtype vs gene knockout Arabidopsis lines. We have grown seedlings for 6 days under 16 hour white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.

About the Experimenter

Name:Dr Felix Jaffe
Head of Lab Name:Dr Matthew Terry
Lab:
Address:School of Biological Sciences
University of Southampton
Bassett Crescent East
Southampton
Postcode: SO16 7PX
Country: UK
 
Telephone Number: 02380594297
Fax Number: 02380594459

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

About this Experiment

Experiment Type: genetic_modification_design
Number of Slides:6
 
Experimental Parameters:
Quality Control Measures Taken:
no-plants-pooled
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Jaffe-temp

Slide: Jaffe_1-5_fr1fr2SA_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Jaffe_1-5_fr1fr2SA_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: Jaffe_1-5_fr1fr2SA_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: fr1fr2SA
Stock Code:
Genetic Background: Col-8
Age: 6
Growth Conditions:
StratificationSeed were sterilised plated on 1/2 MS plates and placed in the dark at 4 oC for 48 hours.
Sterilisation1) Place seed in tube and add 70% Ethanol for 1 min 2) Replace with 10% bleach and treat for 10 min 3) Remove bleach and wash 5 times in sterile filter water 4) Finally re-suspend seed in 200-500 ul Sterile water 5) Seeds are ready for plating out on ½ MS agar
ProtocolPlates containing stratified seed were germinated and grown by transfer to a white light cabinet at 130 μmol/m2/sec for 6 days. Light growth cycle was 16 hours white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
Percentage Agrose10
Substrate Sterilising ProcedureSterilisation of media was by autoclaving for 15 min at 121 oC 15 psi
Plant Spacingclumps of about 20 seedling
Temperature22 °C average, 22 °C day, 22 °C night
Humidity30 % average, 30 % day, 30 % night
MediumMurashige & Skoog basal salt mixture. Modifications: pH 5.7
Lighting(Source: Cool white fluorescent manufactured by Percieval cabinet. Intensity: 130µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.0
Genetic Variation: T-DNA knock out mutation
Tissue: whole plant
Additional Organism Information:
Sample DescriptionRNA was extracted from whole seedlings (Cotyledons, hypocotyl and root)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

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:1.146472096443
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:
RawQ2.08
NoiseAvg:1.96,Stdev:0.04,Max:2.1,Min:1.8
Central-Avg:5908,Count:9
Corner+Avg:55,Count:32
Corner-Avg:5971,Count:32
BackgroundAvg:52.94,Stdev:0.36,Max:53.7,Min:51.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.146472096443
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Jaffe_1-6_fr1fr2SA_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 ("Jaffe_1-6_fr1fr2SA_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: Jaffe_1-6_fr1fr2SA_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: fr1fr2SA
Stock Code:
Genetic Background: Col-8
Age: 6
Growth Conditions:
StratificationSeed were sterilised plated on 1/2 MS plates and placed in the dark at 4 oC for 48 hours.
Sterilisation1) Place seed in tube and add 70% Ethanol for 1 min 2) Replace with 10% bleach and treat for 10 min 3) Remove bleach and wash 5 times in sterile filter water 4) Finally re-suspend seed in 200-500 ul Sterile water 5) Seeds are ready for plating out on ½ MS agar
ProtocolPlates containing stratified seed were germinated and grown by transfer to a white light cabinet at 130 μmol/m2/sec for 6 days. Light growth cycle was 16 hours white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
Percentage Agrose10
Substrate Sterilising ProcedureSterilisation of media was by autoclaving for 15 min at 121 oC 15 psi
Plant Spacingclumps of about 20 seedling
Temperature22 °C average, 22 °C day, 22 °C night
Humidity30 % average, 30 % day, 30 % night
MediumMurashige & Skoog basal salt mixture. Modifications: pH 5.7
Lighting(Source: Cool white fluorescent manufactured by Percieval cabinet. Intensity: 130µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.0
Genetic Variation: T-DNA knock out mutation
Tissue: whole plant
Additional Organism Information:
Sample DescriptionRNA was extracted from whole seedlings (Cotyledons, hypocotyl and root)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:2.335818529129
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.78
NoiseAvg:1.58,Stdev:0.05,Max:1.7,Min:1.5
Central-Avg:4731,Count:9
Corner+Avg:54,Count:32
Corner-Avg:6222,Count:32
BackgroundAvg:46.44,Stdev:0.44,Max:47.7,Min:45.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.335818529129
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Jaffe_1-4_Wt-Col_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 ("Wt Col 4A") 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: Wt Col 4A
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: N60000
Genetic Background: Col-0 (Columbia)
Age: 6
Growth Conditions:
StratificationSeed were sterilised plated on 1/2 MS plates and placed in the dark at 4 oC for 48 hours.
Sterilisation1) Place seed in tube and add 70% Ethanol for 1 min 2) Replace with 10% bleach and treat for 10 min 3) Remove bleach and wash 5 times in sterile filter water 4) Finally re-suspend seed in 200-500 ul Sterile water 5) Seeds are ready for plating out on ½ MS agar
ProtocolPlates containing stratified seed were germinated and grown by transfer to a white light cabinet at 130 μmol/m2/sec for 6 days. Light growth cycle was 16 hours white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
Percentage Agrose10
Substrate Sterilising ProcedureSterilisation of media was by autoclaving for 15 min at 121 oC 15 psi
Plant Spacingclumps of about 20 seedling
Temperature22 °C average, 22 °C day, 22 °C night
Humidity30 % average, 30 % day, 30 % night
MediumMurashige & Skoog basal salt mixture. Modifications: pH 5.7
Lighting(Source: Cool white fluorescent manufactured by Percieval cabinet. Intensity: 130µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.0
Genetic Variation: ecotype
Tissue: whole plant
Additional Organism Information:
Sample DescriptionRNA was extracted from whole seedlings (Cotyledons, hypocotyl and root)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

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:1.270637512207
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:2.02,Stdev:0.04,Max:2.2,Min:1.9
Central-Avg:7089,Count:9
Corner+Avg:63,Count:32
Corner-Avg:7510,Count:32
BackgroundAvg:53.66,Stdev:0.23,Max:54.1,Min:53.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.270637512207
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Jaffe_1-3_Wt-Col_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 ("Wt Col 3A") 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: Wt Col 3A
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: N60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationSeed were sterilised plated on 1/2 MS plates and placed in the dark at 4 oC for 48 hours.
Sterilisation1) Place seed in tube and add 70% Ethanol for 1 min 2) Replace with 10% bleach and treat for 10 min 3) Remove bleach and wash 5 times in sterile filter water 4) Finally re-suspend seed in 200-500 μl Sterile water 5) Seeds are ready for plating out on ½ MS agar
ProtocolPlates containing stratified seed were germinated and grown by transfer to a white light cabinet at 130 μmol/m2/sec for 6 days. Light growth cycle was 16 hours white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
Percentage Agrose10
Substrate Sterilising ProcedureSterilisation of media was by autoclaving for 15 min at 121 oC 15 psi
Plant Spacingclumps of about 20 seedling
Temperature22 °C average, 22 °C day, 22 °C night
Humidity30 % average, 30 % day, 30 % night
MediumMurashige & Skoog basal salt mixture. Modifications: pH 5.7
Lighting(Source: Cool white fluorescent manufactured by Percieval cabinet. Intensity: 130µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Genetic Variation: ecotype
Tissue: whole plant
Additional Organism Information:
Sample DescriptionRNA was extracted from whole seedlings (Cotyledons, hypocotyl and root)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

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:1.263730406761
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:2.01,Stdev:0.07,Max:2.3,Min:1.8
Central-Avg:6663,Count:9
Corner+Avg:63,Count:32
Corner-Avg:7411,Count:32
BackgroundAvg:53.69,Stdev:0.19,Max:54.2,Min:53.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.263730406761
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Hybridisation Set: Jaffe: Expression comparison for white light grown Wt vs knockout arabidopsis_genome

Slide: Jaffe_1-1_Wt-Col_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 ("Jaffe_1-1_Wt-Col_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: Jaffe_1-1_Wt-Col_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-8 (Columbia)
Stock Code: N60000
Genetic Background: Col-0 (Columbia)
Growth Conditions:
StratificationSeed were sterilised plated on 1/2 MS plates and placed in the dark at 4 oC for 48 hours.
Sterilisation1) Place seed in tube and add 70% Ethanol for 1 min 2) Replace with 10% bleach and treat for 10 min 3) Remove bleach and wash 5 times in sterile filter water 4) Finally re-suspend seed in 200-500 μl Sterile water 5) Seeds are ready for plating out on ½ MS agar
ProtocolPlates containing stratified seed were germinated and grown by transfer to a white light cabinet at 130 μmol/m2/sec for 6 days. Light growth cycle was 16 hours white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
Percentage Agrose0.8
Substrate Sterilising ProcedureSterilisation of media was by autoclaving for 15 min at 121 oC 15 ps1
Plant Spacingclumps of about 20 seedling
Temperature22 °C average, 22 °C day, 22 °C night
Humidity30 % average, 30 % day, 30 % night
MediumMurashige & Skoog basal salt mixture. Modifications: pH5.7 with KOH . Applied: N/A
Lighting(Source: Cool white fluorescent manufactured by Percieval. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.0
Genetic Variation: ecotype
Tissue: whole plant
Additional Organism Information:
Sample DescriptionRNA was extracted from whole seedlings (Cotyledons, hypocotyl and root)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

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:1.871939063072
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:
RawQ1.74
NoiseAvg:1.62,Stdev:0.04,Max:1.8,Min:1.5
Central-Avg:10168,Count:9
Corner+Avg:90,Count:32
Corner-Avg:10850,Count:32
BackgroundAvg:41.75,Stdev:0.28,Max:42.2,Min:40.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.871939063072
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Jaffe_1-2_fr1fr2SA_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 ("Jaffe_1-2_fr1fr2SA_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: Jaffe_1-2_fr1fr2SA_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: fr1fr2SA
Stock Code:
Genetic Background: Col-8
Age: 6
Growth Conditions:
StratificationSeed were sterilised plated on 1/2 MS plates and placed in the dark at 4 oC for 48 hours.
Sterilisation1) Place seed in tube and add 70% Ethanol for 1 min 2) Replace with 10% bleach and treat for 10 min 3) Remove bleach and wash 5 times in sterile filter water 4) Finally re-suspend seed in 200-500 μl Sterile water 5) Seeds are ready for plating out on ½ MS agar
ProtocolPlates containing stratified seed were germinated and grown by transfer to a white light cabinet at 130 umol/m2/sec for 6 days. Light growth cycle was 16 hours white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
Percentage Agrose0.8
Substrate Sterilising ProcedureSterilisation of media was by autoclaving for 15 min at 121 oC 15 ps1
Plant Spacingclumps of about 20 seedling
Temperature22 °C average, 22 °C day, 22 °C night
Humidity30 % average, 30 % day, 30 % night
MediumMurashige & Skoog basal salt mixture. Modifications: pH5.7 with KOH . Applied: N/A
Lighting(Source: Cool white fluorescent manufactured by Percieval. Intensity: 130µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.0
Genetic Variation: T-DNA knock out mutation in
Tissue: whole plant
Additional Organism Information:
Sample DescriptionRNA was extracted from whole seedlings (Cotyledons, hypocotyl and root)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

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:1.379349112511
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:
RawQ2.08
NoiseAvg:1.98,Stdev:0.07,Max:2.2,Min:1.8
Central-Avg:10294,Count:9
Corner+Avg:83,Count:32
Corner-Avg:10701,Count:32
BackgroundAvg:48.29,Stdev:0.76,Max:51.0,Min:47.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.379349112511
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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