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Experiment: Expression changes during polyploidisation

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-615

Polyploidisation is frequent in higher plants and contributed significantly to their evolution and adaptation. The sucess of polyploid plants seems to be due to diversification that is least in part generated by major changes in genome organisation and epigenetic regulation. The aim of this experiment is to analyse changes in global gene-expression occurring after autopolyploidisation of an Arabidopsis thaliana line carrying a HPT transgene.Mittelsten Scheid O. et al., PNAS, 1996Mittelsten Scheid O. et al., Nature Genetics, 2003

About the Experimenter

Name: Tuncay Baubec
Head of Lab Name:Dr Ortrun Mittelsten Scheid
Lab:
Address:Gregor Mendel Institute
Dr. Bohrgasse 3
1030 Vienna
Postcode: 1030
Country: Austria
 

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

About this Experiment

Experiment Type: genetic_modification_design
Number of Slides:6
 
Experimental Parameters:
parametergene_knock_out
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Baubec: Expression changes during polyploidisation

Slide: Baubec_615-1_diploid-lineC_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 ("Baubec_615-1_diploid-lineC_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: Baubec_615-1_diploid-lineC_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: line C
Stock Code:
Age: 17
Growth Conditions:
StratificationSeeds were dried after harvesting and cold-treated for minimum 7 days at 4°C
Sterilisation6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying.
ProtocolCold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 25 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in growth chambers at 16h light / 8h dark cycles and 21°C.
Percentage Agrose8
Plant Spacing25
Temperature21 °C average, 21 °C day, 21 °C night
Humidity100 % average, 100 % day, 100 % night
MediumOwn medium: MS macro 50ml B5 micro 1ml ammonium iron citrate 50mg sucrose 10g MES 350mg 8% Agar 450ml
Lighting(Source: Fluorescent manufactured by Phillips. Intensity: 105µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.04
Genetic Variation: knock out mutation in HPT resistance marker
Tissue: whole plant
Additional Organism Information:
Sample DescriptionSample was derived from a pool of 25 17-day old seedlings with no apparent phenotype
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:1.388272404671
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:1.75,Stdev:0.06,Max:1.9,Min:1.6
Central-Avg:7621,Count:9
Corner+Avg:74,Count:32
Corner-Avg:9624,Count:32
BackgroundAvg:42.69,Stdev:0.19,Max:43.1,Min:42.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.388272404671
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Baubec_615-2_diploid-lineC_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 ("Baubec_615-2_diploid-lineC_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: Baubec_615-2_diploid-lineC_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: line C
Stock Code:
Age: 17
Growth Conditions:
StratificationSeeds were dried after harvesting and cold-treated for minimum 7 days at 4°C
Sterilisation6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying.
ProtocolCold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 25 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in growth chambers at 16h light / 8h dark cycles and 21°C.
Percentage Agrose8
Plant Spacing25
Temperature21 °C average, 21 °C day, 21 °C night
Humidity100 % average, 100 % day, 100 % night
MediumOwn medium: MS macro 50ml B5 micro 1ml ammonium iron citrate 50mg sucrose 10g MES 350mg 8% Agar 450ml
Lighting(Source: Fluorescent manufactured by Phillips. Intensity: 105µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.04
Genetic Variation: knock out mutation in HPT resistance marker
Tissue: whole plant
Additional Organism Information:
Sample DescriptionSample was derived from a pool of 25 17-day old seedlings with no apparent phenotype
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:1.132270812988
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:1.80,Stdev:0.07,Max:1.9,Min:1.5
Central-Avg:7740,Count:9
Corner+Avg:76,Count:32
Corner-Avg:9718,Count:32
BackgroundAvg:43.81,Stdev:0.17,Max:44.2,Min:43.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.132270812988
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Baubec_615-3_diploid-lineC_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 ("Baubec_615-3_diploid-lineC_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: Baubec_615-3_diploid-lineC_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: line C
Stock Code:
Age: 17
Growth Conditions:
StratificationSeeds were dried after harvesting and cold-treated for minimum 7 days at 4°C
Sterilisation6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying.
ProtocolCold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 25 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in growth chambers at 16h light / 8h dark cycles and 21°C.
Percentage Agrose8
Plant Spacing25
Temperature21 °C average, 21 °C day, 21 °C night
Humidity100 % average, 100 % day, 100 % night
MediumOwn medium: MS macro 50ml B5 micro 1ml ammonium iron citrate 50mg sucrose 10g MES 350mg 8% Agar 450ml
Lighting(Source: Fluorescent manufactured by Phillips. Intensity: 105µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.04
Genetic Variation: knock out mutation in HPT resistance marker
Tissue: whole plant
Additional Organism Information:
Sample DescriptionSample was derived from a pool of 25 17-day old seedlings with no apparent phenotype
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:1.210028648376
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:
RawQ2.00
NoiseAvg:1.84,Stdev:0.05,Max:2.0,Min:1.7
Central-Avg:8020,Count:9
Corner+Avg:74,Count:32
Corner-Avg:9610,Count:32
BackgroundAvg:45.47,Stdev:0.51,Max:46.4,Min:43.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.210028648376
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Baubec_615-4_tetraploid-lineC_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 ("Baubec_615-4_tetraploid-lineC_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: Baubec_615-4_tetraploid-lineC_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: C4S1
Stock Code:
Age: 17
Growth Conditions:
StratificationSeeds were dried after harvesting and cold-treated for minimum 7 days at 4°C
Sterilisation6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying.
ProtocolCold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 25 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in growth chambers at 16h light / 8h dark cycles and 21°C.
Percentage Agrose8
Plant Spacing25
Temperature21 °C average, 21 °C day, 21 °C night
Humidity100 % average, 100 % day, 100 % night
MediumOwn medium: MS macro 50ml B5 micro 1ml ammonium iron citrate 50mg sucrose 10g MES 350mg 8% Agar 450ml
Lighting(Source: Fluorescent manufactured by Phillips. Intensity: 105µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.04
Genetic Variation: knock out mutation in HPT resistance marker
Tissue: whole plant
Additional Organism Information:
Sample DescriptionSample was derived from a pool of 25 17-day old seedlings with no apparent phenotype
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:1.470324158669
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:1.94,Stdev:0.06,Max:2.1,Min:1.8
Central-Avg:9212,Count:9
Corner+Avg:82,Count:32
Corner-Avg:10856,Count:32
BackgroundAvg:47.29,Stdev:0.21,Max:47.7,Min:46.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.470324158669
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Baubec_615-5_tetraploid-lineC_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 ("Baubec_615-5_tetraploid-lineC_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: Baubec_615-5_tetraploid-lineC_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: C4S1
Stock Code:
Age: 17
Growth Conditions:
StratificationSeeds were dried after harvesting and cold-treated for minimum 7 days at 4°C
Sterilisation6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying.
ProtocolCold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 25 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in growth chambers at 16h light / 8h dark cycles and 21°C.
Percentage Agrose8
Plant Spacing25
Temperature21 °C average, 21 °C day, 21 °C night
Humidity100 % average, 100 % day, 100 % night
MediumOwn medium: MS macro 50ml B5 micro 1ml ammonium iron citrate 50mg sucrose 10g MES 350mg 8% Agar 450ml
Lighting(Source: Fluorescent manufactured by Phillips. Intensity: 105µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.04
Genetic Variation: knock out mutation in HPT resistance marker
Tissue: whole plant
Additional Organism Information:
Sample DescriptionSample was derived from a pool of 25 17-day old seedlings with no apparent phenotype
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.893165290356
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:2.22,Stdev:0.16,Max:3.1,Min:1.9
Central-Avg:9539,Count:9
Corner+Avg:91,Count:32
Corner-Avg:11132,Count:32
BackgroundAvg:50.50,Stdev:0.35,Max:51.3,Min:49.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.893165290356
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Baubec_615-6_tetraploid-lineC_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 ("Baubec_615-6_tetraploid-lineC_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: Baubec_615-6_tetraploid-lineC_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: C4S1
Stock Code:
Age: 17
Growth Conditions:
StratificationSeeds were dried after harvesting and cold-treated for minimum 7 days at 4°C
Sterilisation6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying.
ProtocolCold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 25 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in growth chambers at 16h light / 8h dark cycles and 21°C.
Percentage Agrose8
Plant Spacing25
Temperature21 °C average, 21 °C day, 21 °C night
Humidity100 % average, 100 % day, 100 % night
MediumOwn medium: MS macro 50ml B5 micro 1ml ammonium iron citrate 50mg sucrose 10g MES 350mg 8% Agar 450ml
Lighting(Source: Fluorescent manufactured by Phillips. Intensity: 105µEinsteins. Wavelength: White)
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.04
Genetic Variation: knock out mutation in HPT resistance marker
Tissue: whole plant
Additional Organism Information:
Sample DescriptionSample was derived from a pool of 25 17-day old seedlings with no apparent phenotype
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.768997430801
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:
RawQ2.18
NoiseAvg:2.25,Stdev:0.05,Max:2.4,Min:2.1
Central-Avg:9704,Count:9
Corner+Avg:85,Count:32
Corner-Avg:10613,Count:32
BackgroundAvg:51.02,Stdev:0.36,Max:51.9,Min:49.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.768997430801
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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