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Experiment: Effect of the explosive compound TNT on wild type sedlings

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-472

The aim of this experiment was to identify the genes involved in the detoxification of the toxic pollutant and explosive compound 2,4,6-trinitrotolune (TNT).Fourteen-day-old, liquid culture grown, Arabidopsis seedlings, ecotype Col0 (NASC stock code N1093), were dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF) solvent, or 60 ul DMF only. After six hours, RNA was extracted and used for the microarray analysis.Further details and characterisation of glucosyltransferases identified using this method are presented in: Gandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases (2008). The Plant Journal Dec;56(6):963-74.

About the Experimenter

Name:Dr Elizabeth Rylott
Head of Lab Name:Prof Neil Bruce
Lab:
Address:CNAP
Department of Biology
University of York
PO Box 373
York
Postcode: YO10 5YW
Country: UK
 
Telephone Number: 01904 328777
Fax Number: 01904 328801

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:6
 
Experimental Parameters:
parametercompound_based_treatment
Quality Control Measures Taken:
References:
 
Other Information:
GEO accessionGSE18983

Slides in this Experiment

Hybridisation Set: Rylott: Effect of the explosive compound TNT on wild type sedlings_genome

Slide: Rylott_1-4_DMF-treated-control-seedlings_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 ("Rylott_1-4_DMF-treated-control-seedlings_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: Rylott_1-4_DMF-treated-control-seedlings_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1093
Growth Conditions:
Stratificationseeds were stratified for three days at 4 ºC in the dark on agar plates containing half strength Murashige and Skoog medium with 20 mM sucrose.
SterilizationSeeds were sterilized using Conclor disinfectant tablets. One tablet was dissolved in 14 ml of dH20 with 2-3 drops of 1% Tween 20, then 5 ml of this solution added to 45 ml of 95% ethanol. Any precipitate was allowed to settle, then 1 ml of the ethanol/ Conchlor solution was added to each Eppendorf tube of seeds. The tubes were shaken for five minutes then the Conchlor solution removed and the seeds washed six times with 95% ethanol and the seeds air-dried.
Growth protocolSeeds on plates were transferred to 125 uM.m-1.s-2 light, 16 h photoperiod, at 20 - 18 oC day and night temperature for 24 h, then ten seedlings transferred to a 500 ml conical flask containing 100 ml 1/2MS medium, 20 mM sucrose and 1 x Gamborg?s vitamin solution with 130 rpm, 20 uM.m-1.s-2 light for 13 days. The flask was dosed with 60 ul dimethyl formamide (DMF). After 6 h, RNA was extracted.
ReferenceGandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases 2008, The Plant Journal (In Press).
LocationGrowth Room
Growth substrate typeAgar
Average temperature20ºC
Average humidity75%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modification20 mM Sucrose (pre autoclave) and 1 x filter-sterilized Gamborg's vitamin solution (post autoclave)added
Developmental Stage:
Developmental stage(Source: Boyes Key: Paradigm Genetics)1.05
Tissue: whole plant
in vivo Treatment:
TreatmentSeedlings in flasks were dosed with 60 microlitres dimethyl formamide (DMF)solvent
Other Information:
sampledescriptionWhole, 14-day-old seedlings

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:0.520563
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:
RawQ1.88
NoiseAvg:2.09,Stdev:0.09,Max:2.3,Min:1.9
BackgroundAvg:46.88,Stdev:0.89,Max:49.1,Min:44.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.520563
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Rylott_1-3_TNT-treated-seedlings_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 ("Rylott_1-3_TNT-treated-seedlings_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: Rylott_1-3_TNT-treated-seedlings_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1093
Growth Conditions:
Stratificationseeds were stratified for three days at 4 ºC in the dark on agar plates containing half strength Murashige and Skoog medium with 20 mM sucrose.
SterilizationSeeds were sterilized using Conclor disinfectant tablets. One tablet was dissolved in 14 ml of dH20 with 2-3 drops of 1% Tween 20, then 5 ml of this solution added to 45 ml of 95% ethanol. Any precipitate was allowed to settle, then 1 ml of the ethanol/ Conchlor solution was added to each Eppendorf tube of seeds. The tubes were shaken for five minutes then the Conchlor solution removed and the seeds washed six times with 95% ethanol and the seeds air-dried.
Growth protocolSeeds on plates were transferred to 125 uM.m-1.s-2 light, 16 h photoperiod, at 20 - 18 oC day and night temperature for 24 h, then ten seedlings transferred to a 500 ml conical flask containing 100 ml 1/2MS medium, 20 mM sucrose and 1 x Gamborg's vitamin solution with 130 rpm, 20 uM.m-1.s-2 light for 13 days. The flask was dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF). After 6 h, RNA was extracted.
ReferenceGandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases 2008, The Plant Journal (In Press).
LocationGrowth Room
Growth substrate typeAgar
Average temperature20ºC
Average humidity75%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modification20 mM Sucrose (pre autoclave) and 1 x filter-sterilized Gamborg's vitamin solution (post autoclave)added
Developmental Stage:
Developmental stage(Source: Boyes Key: Paradigm Genetics)1.05
Tissue: whole plant
in vivo Treatment:
TreatmentSeedlings in flasks were dosed with 60 micromolar TNT dissolved in 60 microlitres dimethyl formamide (DMF)solvent
Other Information:
sampledescriptionWhole, 14-day-old seedlings

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:0.732456
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:
RawQ1.98
NoiseAvg:2.04,Stdev:0.05,Max:2.1,Min:1.9
BackgroundAvg:50.18,Stdev:0.81,Max:52.0,Min:48.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.732456
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Rylott_1-2_TNT-treated-seedlings_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Rylott_1-2_TNT-treated-seedlings_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: Rylott_1-2_TNT-treated-seedlings_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1093
Growth Conditions:
Stratificationseeds were stratified for three days at 4 ºC in the dark on agar plates containing half strength Murashige and Skoog medium with 20 mM sucrose.
SterilizationSeeds were sterilized using Conclor disinfectant tablets. One tablet was dissolved in 14 ml of dH20 with 2-3 drops of 1% Tween 20, then 5 ml of this solution added to 45 ml of 95% ethanol. Any precipitate was allowed to settle, then 1 ml of the ethanol/ Conchlor solution was added to each Eppendorf tube of seeds. The tubes were shaken for five minutes then the Conchlor solution removed and the seeds washed six times with 95% ethanol and the seeds air-dried.
Growth protocolSeeds on plates were transferred to 125 uM.m-1.s-2 light, 16 h photoperiod, at 20 - 18 oC day and night temperature for 24 h, then ten seedlings transferred to a 500 ml conical flask containing 100 ml 1/2MS medium, 20 mM sucrose and 1 x Gamborg's vitamin solution with 130 rpm, 20 uM.m-1.s-2 light for 13 days. The flask was dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF). After 6 h, RNA was extracted.
ReferenceGandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases 2008, The Plant Journal (In Press).
LocationGrowth Room
Growth substrate typeAgar
Average temperature20ºC
Average humidity75%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modification20 mM Sucrose (pre autoclave) and 1 x filter-sterilized Gamborg's vitamin solution (post autoclave)added
Developmental Stage:
Developmental stage(Source: Boyes Key: Paradigm Genetics)1.05
Tissue: whole plant
in vivo Treatment:
TreatmentSeedlings in flasks were dosed with 60 micromolar TNT dissolved in 60 microlitres dimethyl formamide (DMF)solvent
Other Information:
sampledescriptionWhole, 14-day-old seedlings

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:0.327868
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:
RawQ2.74
NoiseAvg:5.62,Stdev:0.29,Max:6.3,Min:4.9
BackgroundAvg:77.18,Stdev:2.19,Max:81.7,Min:71.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.327868
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Rylott_1-1_TNT-treated-seedlings_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 ("Rylott_1-1_TNT-treated-seedlings_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: Rylott_1-1_TNT-treated-seedlings_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1093
Growth Conditions:
Stratificationseeds were stratified for three days at 4 ºC in the dark on agar plates containing half strength Murashige and Skoog medium with 20 mM sucrose.
SterilizationSeeds were sterilized using Conclor disinfectant tablets. One tablet was dissolved in 14 ml of dH20 with 2-3 drops of 1% Tween 20, then 5 ml of this solution added to 45 ml of 95% ethanol. Any precipitate was allowed to settle, then 1 ml of the ethanol/ Conchlor solution was added to each Eppendorf tube of seeds. The tubes were shaken for five minutes then the Conchlor solution removed and the seeds washed six times with 95% ethanol and the seeds air-dried.
Growth protocolSeeds on plates were transferred to 125 uM.m-1.s-2 light, 16 h photoperiod, at 20 - 18 oC day and night temperature for 24 h, then ten seedlings transferred to a 500 ml conical flask containing 100 ml 1/2MS medium, 20 mM sucrose and 1 x Gamborg's vitamin solution with 130 rpm, 20 uM.m-1.s-2 light for 13 days. The flask was dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF). After 6 h, RNA was extracted.
ReferenceGandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases 2008, The Plant Journal (In Press).
LocationGrowth Room
Growth substrate typeAgar
Average temperature20ºC
Average humidity75%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modification20 mM Sucrose (pre autoclave) and 1 x filter-sterilized Gamborg's vitamin solution (post autoclave)added
Developmental Stage:
Tissue: whole plant
in vivo Treatment:
TreatmentSeedlings in flasks were dosed with 60 micromolar TNT dissolved in 60 microlitres dimethyl formamide (DMF)solvent
Other Information:
sampledescriptionWhole, 14-day-old seedlings

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:0.514286
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:
RawQ1.89
NoiseAvg:2.14,Stdev:0.11,Max:2.6,Min:1.9
BackgroundAvg:46.03,Stdev:1.06,Max:49.1,Min:43.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.514286
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Rylott_1-5_DMF-treated-control-seedlings_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 ("Rylott_1-5_DMF-treated-control-seedlings_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: Rylott_1-5_DMF-treated-control-seedlings_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1093
Growth Conditions:
Stratificationseeds were stratified for three days at 4 ºC in the dark on agar plates containing half strength Murashige and Skoog medium with 20 mM sucrose.
SterilizationSeeds were sterilized using Conclor disinfectant tablets. One tablet was dissolved in 14 ml of dH20 with 2-3 drops of 1% Tween 20, then 5 ml of this solution added to 45 ml of 95% ethanol. Any precipitate was allowed to settle, then 1 ml of the ethanol/ Conchlor solution was added to each Eppendorf tube of seeds. The tubes were shaken for five minutes then the Conchlor solution removed and the seeds washed six times with 95% ethanol and the seeds air-dried.
Growth protocolSeeds on plates were transferred to 125 uM.m-1.s-2 light, 16 h photoperiod, at 20 - 18 oC day and night temperature for 24 h, then ten seedlings transferred to a 500 ml conical flask containing 100 ml 1/2MS medium, 20 mM sucrose and 1 x Gamborg?s vitamin solution with 130 rpm, 20 uM.m-1.s-2 light for 13 days. The flask was dosed with 60 ul dimethyl formamide (DMF). After 6 h, RNA was extracted.
ReferenceGandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases 2008, The Plant Journal (In Press).
LocationGrowth Room
Growth substrate typeAgar
Average temperature20ºC
Average humidity75%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modification20 mM Sucrose (pre autoclave) and 1 x filter-sterilized Gamborg's vitamin solution (post autoclave)added
Developmental Stage:
Developmental stage(Source: Boyes Key: Paradigm Genetics)1.05
Tissue: whole plant
in vivo Treatment:
TreatmentSeedlings in flasks were dosed with 60 microlitres dimethyl formamide (DMF)solvent
Other Information:
sampledescriptionWhole, 14-day-old seedlings

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:0.400926
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:
RawQ2.63
NoiseAvg:5.32,Stdev:0.29,Max:6.3,Min:4.5
BackgroundAvg:68.62,Stdev:2.05,Max:73.5,Min:63.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.400926
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Rylott_1-6_DMF-treated-control-seedlings_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 ("Rylott_1-6_DMF-treated-control-seedlings_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: Rylott_1-6_DMF-treated-control-seedlings_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1093
Growth Conditions:
Stratificationseeds were stratified for three days at 4 ºC in the dark on agar plates containing half strength Murashige and Skoog medium with 20 mM sucrose.
SterilizationSeeds were sterilized using Conclor disinfectant tablets. One tablet was dissolved in 14 ml of dH20 with 2-3 drops of 1% Tween 20, then 5 ml of this solution added to 45 ml of 95% ethanol. Any precipitate was allowed to settle, then 1 ml of the ethanol/ Conchlor solution was added to each Eppendorf tube of seeds. The tubes were shaken for five minutes then the Conchlor solution removed and the seeds washed six times with 95% ethanol and the seeds air-dried.
Growth protocolSeeds on plates were transferred to 125 uM.m-1.s-2 light, 16 h photoperiod, at 20 - 18 oC day and night temperature for 24 h, then ten seedlings transferred to a 500 ml conical flask containing 100 ml 1/2MS medium, 20 mM sucrose and 1 x Gamborg?s vitamin solution with 130 rpm, 20 uM.m-1.s-2 light for 13 days. The flask was dosed with 60 ul dimethyl formamide (DMF). After 6 h, RNA was extracted.
ReferenceGandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases 2008, The Plant Journal (In Press).
LocationGrowth Room
Growth substrate typeAgar
Average temperature20ºC
Average humidity75%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modification20 mM Sucrose (pre autoclave) and 1 x filter-sterilized Gamborg's vitamin solution (post autoclave)added
Developmental Stage:
Developmental stage(Source: Boyes Key: Paradigm Genetics)1.05
Tissue: whole plant
in vivo Treatment:
TreatmentSeedlings in flasks were dosed with 60 microlitres dimethyl formamide (DMF)solvent
Other Information:
sampledescriptionWhole, 14-day-old seedlings

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:0.609097
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:
RawQ1.86
NoiseAvg:2.05,Stdev:0.14,Max:2.6,Min:1.8
BackgroundAvg:47.04,Stdev:0.77,Max:48.9,Min:45.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.609097
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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