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Experiment: Identification of AtATM dependent X-ray inducible transcript

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-370

ATM plays an important role in the response of plants to forms of DNA damage that cause DNA double strand breaks. Arabidopsis ATM has been shown to be important for the transcriptional induction of several DNA repair genes including AtRAD51 (Garcia et al 2003 Plant Cell 15, 119-132). In our experiment we compare the transcript profiles of wild type and mutant plants exposed to 10Gy X-rays (dose rate ~0.8 Gy/min). Plants will be grown on 0.5*MS + 1%sucrose at 22degC under short (9h) day conditions and treated after 5h light. We will irradiate wild type (Col-0) and atatm-3 mutant (N589805) seedlings at growth stage 1.06 and harvest tissue 1h post irradiation. RNA will be isolated using the SV RNA isolation kit (Promega). Comparison of the transcript profiles will then allow AtATM-dependent X-ray inducible transcripts to be identified.

About the Experimenter

Name:Dr Chris West
Head of Lab Name:Dr Chris West
Lab:
Institute: University of Leeds
Address:Centre for Plant Sciences
University of Leeds
Woodhouse Lane
Leeds
West Yorkshire
Postcode: LS2 9JT
Country: UK
 
Telephone Number: 01133432915
Fax Number: 0113 3433144

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

About this Experiment

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

Slides in this Experiment

Hybridisation Set: West: Identification of AtATM dependent X-ray inducible transcript_genome

Slide: West_2-1_Col0_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 ("West_2-1_Col0_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: West_2-1_Col0_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1092
Growth Conditions:
stratificationOvernight 4ºC in dark
sterilisation10 minutes soaking in sterilisation solution (10 % (v/v) house hold bleach, 1 % (v/v) Triton X-100) followed by 5 repeat rinses of sd H2O
protocolSeedlings grown on 0.5 x MS media containing 1 % phytoagar and 1 % sucrose in a growth room with the following conditions: 9/15 hrs light/dark, 22°C, 65 µEm-2s-1
LocationGrowth Room
Growth substratePhytagar
SterilisationStandard media autoclave
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Developmental stage(Source: Boyes Key)1.04
Tissue: whole plant
in vivo Treatment:
treatmentFourteen day old seedlings were exposed, whilst on the plates, to a 10 Gy dose of X-rays over the course of 7.5 minutes using a 320 kV X-ray system. Plants were harvested 1 hour following treatment.
Additional Organism Information:
sampledescriptionWhole seedlings (14 days old)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Promega SV Total RNA isolation kit
Method:
ProtocolAs per manufacturer's instructions
Type: total RNA

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:1.157894
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:
RawQ1.93
NoiseAvg:2.39,Stdev:0.06,Max:2.7,Min:2.3
BackgroundAvg:49.71,Stdev:0.50,Max:51.3,Min:48.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.157894
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: West_2-2_Col0_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 ("West_2-2_Col0_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: West_2-2_Col0_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1092
Growth Conditions:
stratificationOvernight 4ºC in dark
sterilisation10 minutes soaking in sterilisation solution (10 % (v/v) house hold bleach, 1 % (v/v) Triton X-100) followed by 5 repeat rinses of sd H2O
protocolSeedlings grown on 0.5 x MS media containing 1 % phytoagar and 1 % sucrose in a growth room with the following conditions: 9/15 hrs light/dark, 22°C, 65 µEm-2s-1
LocationGrowth Room
Growth substratePhytagar
SterilisationStandard media autoclave
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Developmental stage(Source: Boyes Key)1.04
Tissue: whole plant
in vivo Treatment:
treatmentFourteen day old seedlings were exposed, whilst on the plates, to a 10 Gy dose of X-rays over the course of 7.5 minutes using a 320 kV X-ray system. Plants were harvested 1 hour following treatment.
Additional Organism Information:
sampledescriptionWhole seedlings (14 days old)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Promega SV Total RNA isolation kit
Method:
ProtocolAs per manufacturer's instructions
Type: total RNA

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:1.080651
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:
RawQ2.36
NoiseAvg:3.15,Stdev:0.50,Max:6.1,Min:2.7
BackgroundAvg:64.20,Stdev:0.50,Max:65.6,Min:63.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.080651
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: West_2-3_Col0_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 ("West_2-3_Col0_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: West_2-3_Col0_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col0
Stock Code: N1092
Growth Conditions:
stratificationOvernight 4ºC in dark
sterilisation10 minutes soaking in sterilisation solution (10 % (v/v) house hold bleach, 1 % (v/v) Triton X-100) followed by 5 repeat rinses of sd H2O
protocolSeedlings grown on 0.5 x MS media containing 1 % phytoagar and 1 % sucrose in a growth room with the following conditions: 9/15 hrs light/dark, 22°C, 65 µEm-2s-1
LocationGrowth Room
Growth substratePhytagar
SterilisationStandard media autoclave
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Developmental stage(Source: Boyes Key)1.04
Tissue: whole plant
in vivo Treatment:
treatmentFourteen day old seedlings were exposed, whilst on the plates, to a 10 Gy dose of X-rays over the course of 7.5 minutes using a 320 kV X-ray system. Plants were harvested 1 hour following treatment.
Additional Organism Information:
sampledescriptionWhole seedlings (14 days old)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Promega SV Total RNA isolation kit
Method:
ProtocolAs per manufacturer's instructions
Type: total RNA

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:0.860276
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:
RawQ2.24
NoiseAvg:2.81,Stdev:0.06,Max:3.0,Min:2.5
BackgroundAvg:60.31,Stdev:0.51,Max:62.0,Min:59.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.860276
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: West_2-4_atm_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 ("West_2-4_atm_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: West_2-4_atm_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: ATM1
Stock Code: N589805
Growth Conditions:
stratificationOvernight 4ºC in dark
sterilisation10 minutes soaking in sterilisation solution (10 % (v/v) house hold bleach, 1 % (v/v) Triton X-100) followed by 5 repeat rinses of sd H2O
protocolSeedlings grown on 0.5 x MS media containing 1 % phytoagar and 1 % sucrose in a growth room with the following conditions: 9/15 hrs light/dark, 22°C, 65 µEm-2s-1
LocationGrowth Room
Growth substratePhytagar
SterilisationStandard media autoclave
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Developmental stage(Source: Boyes Key)1.04
Tissue: whole plant
in vivo Treatment:
treatmentFourteen day old seedlings were exposed, whilst on the plates, to a 10 Gy dose of X-rays over the course of 7.5 minutes using a 320 kV X-ray system. Plants were harvested 1 hour following treatment.
Additional Organism Information:
sampledescriptionWhole seedlings (14 days old)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Promega SV Total RNA isolation kit
Method:
ProtocolAs per manufacturer's instructions
Type: total RNA

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:1.099115
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:
RawQ2.60
NoiseAvg:3.13,Stdev:0.09,Max:3.4,Min:2.9
BackgroundAvg:73.19,Stdev:1.13,Max:75.8,Min:71.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.099115
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: West_2-5_atm_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 ("West_2-5_atm_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: West_2-5_atm_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: ATM1
Stock Code: N589805
Growth Conditions:
stratificationOvernight 4ºC in dark
sterilisation10 minutes soaking in sterilisation solution (10 % (v/v) house hold bleach, 1 % (v/v) Triton X-100) followed by 5 repeat rinses of sd H2O
protocolSeedlings grown on 0.5 x MS media containing 1 % phytoagar and 1 % sucrose in a growth room with the following conditions: 9/15 hrs light/dark, 22°C, 65 µEm-2s-1
LocationGrowth Room
Growth substratePhytagar
SterilisationStandard media autoclave
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Developmental stage(Source: Boyes Key)1.04
Tissue: whole plant
in vivo Treatment:
treatmentFourteen day old seedlings were exposed, whilst on the plates, to a 10 Gy dose of X-rays over the course of 7.5 minutes using a 320 kV X-ray system. Plants were harvested 1 hour following treatment.
Additional Organism Information:
sampledescriptionWhole seedlings (14 days old)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Promega SV Total RNA isolation kit
Method:
ProtocolAs per manufacturer's instructions
Type: total RNA

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:1.049264
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:
RawQ2.39
NoiseAvg:2.92,Stdev:0.08,Max:3.1,Min:2.6
BackgroundAvg:63.58,Stdev:0.48,Max:65.0,Min:62.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.049264
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: West_2-6_atm_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 ("West_2-6_atm_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: West_2-6_atm_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: ATM1
Stock Code: N589805
Growth Conditions:
stratificationOvernight 4ºC in dark
sterilisation10 minutes soaking in sterilisation solution (10 % (v/v) house hold bleach, 1 % (v/v) Triton X-100) followed by 5 repeat rinses of sd H2O
protocolSeedlings grown on 0.5 x MS media containing 1 % phytoagar and 1 % sucrose in a growth room with the following conditions: 9/15 hrs light/dark, 22°C, 65 µEm-2s-1
LocationGrowth Room
Growth substratePhytagar
SterilisationStandard media autoclave
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Developmental stage(Source: Boyes Key)1.04
Tissue: whole plant
in vivo Treatment:
treatmentFourteen day old seedlings were exposed, whilst on the plates, to a 10 Gy dose of X-rays over the course of 7.5 minutes using a 320 kV X-ray system. Plants were harvested 1 hour following treatment.
Additional Organism Information:
sampledescriptionWhole seedlings (14 days old)
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Promega SV Total RNA isolation kit
Method:
ProtocolAs per manufacturer's instructions
Type: total RNA

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:1.071886
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.17
NoiseAvg:2.71,Stdev:0.08,Max:3.1,Min:2.5
BackgroundAvg:58.66,Stdev:0.53,Max:59.9,Min:57.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.071886
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team