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Experiment: Inhibitor of auxin response LI5

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-484

LI5 is an inhibitor of auxin response identified in chemical genetics screen for lateral root emergence inhibitors. Chemicals identified in this screen blocked the auxin induction of LAX3 in the outer tissues (Swarup et al, Nat Cell Biol, 2008). We observed that LI5 affected many other auxin responsive genes (unpublished data, November 2008). Our aim is to identified the overall effects on auxin response using the Arabidopsis thaliana chip Ath1. 3 days old Col-0 Arabidopsis seedlings, germinated on NPA 10uM, were treated for 6 hours with [DMSO, LI5 (5uM), NAA (10uM) or LI5 (5uM) + NAA (10uM)], the shoot and root tip removed and RNA was extracted from the mature part of the root.

About the Experimenter

Name:Mr. Antoine Larrieu
Head of Lab Name:Prof Malcolm Bennett
Lab:
Address:University of Nottingham
Sutton Bonington Campus
School of Biosciences
Plant Sciences
Postcode: LE12 5RD
Country: United Kingdom
 
Telephone Number: 00441159516377

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:12
 
Experimental Parameters:
parameter compound_based_treatment
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Larrieu: Inhibitor of auxin response LI5_genome

Slide: Larrieu_1-1_mock-treated_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 ("Larrieu_1-1_mock-treated_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: Larrieu_1-1_mock-treated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21 °C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 0.035% DMSO. DMSO was added to the medium after autoclaving
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.559053480625
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:2.17,Stdev:0.08,Max:2.4,Min:2.0
Central-Avg:9214,Count:9
Corner+Avg:94,Count:32
Corner-Avg:11059,Count:32
BackgroundAvg:47.37,Stdev:0.45,Max:48.3,Min:46.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.559053480625
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-2_mock-treated_Rep2_ATH1

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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 ("Larrieu_1-2_mock-treated_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: Larrieu_1-2_mock-treated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 0.035% DMSO. DMSO was added to the medium after autoclaving
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.558001816273
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:
RawQ2.10
NoiseAvg:2.31,Stdev:0.08,Max:2.5,Min:2.1
Central-Avg:9069,Count:9
Corner+Avg:93,Count:32
Corner-Avg:10640,Count:32
BackgroundAvg:50.16,Stdev:0.50,Max:51.5,Min:49.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.558001816273
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-3_mock-treated_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 ("Larrieu_1-3_mock-treated_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: Larrieu_1-3_mock-treated_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 0.035% DMSO. DMSO was added to the medium after autoclaving
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.500152111053
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:
RawQ2.31
NoiseAvg:2.60,Stdev:0.11,Max:2.9,Min:2.3
Central-Avg:9178,Count:9
Corner+Avg:93,Count:32
Corner-Avg:10570,Count:32
BackgroundAvg:56.08,Stdev:0.50,Max:57.3,Min:54.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.500152111053
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-4_LI5-treated_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 ("Larrieu_1-4_LI5-treated_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: Larrieu_1-4_LI5-treated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 5uM LI5 (LI5 made in house, diluted in DMSO). DMSO final concentration is 0.035%. LI5 and DMSO were added to the medium after autoclaving
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.641309261322
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:2.07,Stdev:0.07,Max:2.2,Min:1.9
Central-Avg:9286,Count:9
Corner+Avg:89,Count:32
Corner-Avg:10980,Count:32
BackgroundAvg:47.54,Stdev:0.42,Max:48.4,Min:45.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.641309261322
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-5_LI5-treated_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 ("Larrieu_1-5_LI5-treated_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: Larrieu_1-5_LI5-treated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 5uM LI5 (LI5 made in house, diluted in DMSO). DMSO final concentration is 0.035%. LI5 and DMSO were added to the medium after autoclaving
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:1.220698833466
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:1.86,Stdev:0.07,Max:2.1,Min:1.7
Central-Avg:6960,Count:9
Corner+Avg:73,Count:32
Corner-Avg:7628,Count:32
BackgroundAvg:43.67,Stdev:0.17,Max:44.1,Min:43.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.220698833466
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-6_LI5-treated_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 ("Larrieu_1-6_LI5-treated_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: Larrieu_1-6_LI5-treated_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 5uM LI5 (LI5 made in house, diluted in DMSO). DMSO final concentration is 0.035%. LI5 and DMSO were added to the medium after autoclaving
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:1.197031974792
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:1.91,Stdev:0.07,Max:2.1,Min:1.7
Central-Avg:7903,Count:9
Corner+Avg:69,Count:32
Corner-Avg:8533,Count:32
BackgroundAvg:46.70,Stdev:0.30,Max:47.5,Min:46.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.197031974792
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-7_Auxin_treated_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 ("Larrieu_1-7_Auxin_treated_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: Larrieu_1-7_Auxin_treated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 10uM NAA (Sigma, UK). NAA was diluted in DMSO. DMSO final concentration was 0.035%. NAA and DMSO were added to the medium after autoclaving.
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.948153376579
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:2.03,Stdev:0.08,Max:2.2,Min:1.8
Central-Avg:7161,Count:9
Corner+Avg:66,Count:32
Corner-Avg:8604,Count:32
BackgroundAvg:47.69,Stdev:0.47,Max:48.7,Min:46.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.948153376579
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-8_Auxin_treated_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 ("Larrieu_1-8_Auxin_treated_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: Larrieu_1-8_Auxin_treated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 10uM NAA (Sigma, UK). NAA was diluted in DMSO. DMSO final concentration was 0.035%. NAA and DMSO were added to the medium after autoclaving.
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.598640978336
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:
RawQ2.22
NoiseAvg:2.35,Stdev:0.07,Max:2.7,Min:2.2
Central-Avg:9295,Count:9
Corner+Avg:86,Count:32
Corner-Avg:9468,Count:32
BackgroundAvg:51.71,Stdev:0.41,Max:53.1,Min:50.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.598640978336
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-9_Auxin_treated_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 ("Larrieu_1-9_Auxin_treated_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: Larrieu_1-9_Auxin_treated_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose and 10uM NAA (Sigma, UK). NAA was diluted in DMSO. DMSO final concentration was 0.035%. NAA and DMSO were added to the medium after autoclaving.
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction.
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.542548596859
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:
RawQ2.35
NoiseAvg:2.57,Stdev:0.06,Max:2.8,Min:2.4
Central-Avg:8234,Count:9
Corner+Avg:78,Count:32
Corner-Avg:9090,Count:32
BackgroundAvg:54.09,Stdev:0.68,Max:55.4,Min:52.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.542548596859
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-10_Auxin-LI5-treated_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 ("Larrieu_1-10_Auxin-LI5-treated_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: Larrieu_1-10_Auxin-LI5-treated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose, 10uM NAA (Sigma, UK) and 5uM LI5 (made in house, diluted in DMSO). NAA was diluted in DMSO. DMSO final concentration was 0.035%. NAA and LI5 were added to the medium after autoclaving.
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.696868181229
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:
RawQ2.04
NoiseAvg:2.04,Stdev:0.08,Max:2.3,Min:1.8
Central-Avg:8171,Count:9
Corner+Avg:82,Count:32
Corner-Avg:9943,Count:32
BackgroundAvg:47.88,Stdev:0.59,Max:49.3,Min:46.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.696868181229
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-11_Auxin-LI5-treated_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 ("Larrieu_1-11_Auxin-LI5-treated_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: Larrieu_1-11_Auxin-LI5-treated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose, 10uM NAA (Sigma, UK) and 5uM LI5 (made in house, diluted in DMSO). NAA was diluted in DMSO. DMSO final concentration was 0.035%. NAA and LI5 were added to the medium after autoclaving.
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.763158082962
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:2.10,Stdev:0.05,Max:2.3,Min:2.0
Central-Avg:8377,Count:9
Corner+Avg:71,Count:32
Corner-Avg:9361,Count:32
BackgroundAvg:50.31,Stdev:0.44,Max:51.4,Min:49.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.763158082962
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Larrieu_1-12_Auxin-LI5-treated_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 ("Larrieu_1-12_Auxin-LI5-treated_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: Larrieu_1-12_Auxin-LI5-treated_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
Growth mediumMurashige & Skoog basal salt mixture
stratificationSeeds were stratified two days at 4 degrees
sterilisationSeeds were surface sterilised 8 minutes in 50% bleach and rinsed 5 times in sterile distilled water.
LocationGrowth Room
Growth substrateBacto-agar
Average humidity100%
Average temperature21°C
Growth protocolArabidopsis Col-0 seeds were sown on plates containing 1/2 strength MS (Murashige and Skoog) salts (Sigma, UK) with 1% sucrose (Fischer Scientific, UK). pH was equilibrated to 5.7 with NaOH. 1% agar (Bacto Agar, Appleton Woods, UK) was added just before autoclaving the medium at 120 degrees for 11 minutes. When the medium had cool down to 60 degrees, NPA (Sigma, UK) was added at a final concentration of 10uM. Seeds were germinated and grown for 5 days under continuous white light (80 uE) at 21°C
Developmental Stage:
Developmental stage(Source: Paradigm Genetics - Boye's Key)1.0
Tissue: Mature root
in vivo Treatment:
Treatment5 days after sowing, seedlings were transferred for 6 hours to agar plates containing 1/2 strength MS (Murashige and Skoog) salts (pH 5.7), with 1% sucrose, 10uM NAA (Sigma, UK) and 5uM LI5 (made in house, diluted in DMSO). NAA was diluted in DMSO. DMSO final concentration was 0.035%. NAA and LI5 were added to the medium after autoclaving.
Additional Organism Information:
Sample descriptionAt the end of the treatment, shoots and root tips were removed and the mature part of the root used for RNA extraction
Separation Technique: Shoot and root tip were cut off using a razor blade.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

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

Labelling Protocol Information

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

Slide Information

Date Hybridised:

Array Design Information

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

Scanning Protocol Information

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

Image Analysis Information

Scaling Factor:0.801166951656
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:0.07,Max:2.4,Min:2.0
Central-Avg:8502,Count:9
Corner+Avg:78,Count:32
Corner-Avg:9213,Count:32
BackgroundAvg:50.74,Stdev:0.44,Max:51.6,Min:49.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.801166951656
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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