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Experiment: Seedling transcriptome affected by a far-red light preconditioning treatment to block chloroplast development.

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-89

This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway.

The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway.

We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment.

In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall.

The proposed experiment will consist of:

wild-type: D-pretreated (x2 biological replicates)

wild-type: FR-preconditioned (x2)

gun1,gun5: D-pretreated (x2)

gun1,gun5: FR-preconditioned (x2)

phyA: D-pretreated (x1)

phyA: FR-preconditioned (x1)

About the Experimenter

Name:Dr Alex McCormac
Head of Lab Name:Dr Matthew Terry
Lab:
Institute: University of Southampton
Address:School of Biological Sciences
Division of Cell Sciences
University of Southampton
Bassett Crescent East
Southampton
Postcode: SO16 7PX
Country: UK
 
Telephone Number: 023 80594297
Fax Number: 023 80594319

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; compound_based_treatment_design
Number of Slides:10
 
Experimental Parameters:
parameter gene_knock_out
parametercompound_based_treatment
Quality Control Measures Taken:
no-plants-pooled300 seedlings
References:
 
Other Information:
ArrayExpressAccessionE-NASC-41

Slides in this Experiment

Hybridisation Set: McCormac_genome_Exp2_Rep1

Slide: McCormac_2-1_wildtype-Frprecon_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 ("McCormac_2-1_wildtype-Frprecon_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: McCormac_2-1_wildtype-Frprecon_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentwild type seedlings pre-conditioned under far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.263873
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:
RawQ3.35
NoiseAvg:5.75,Stdev:0.27,Max:6.5,Min:4.9
BackgroundAvg:84.85,Stdev:1.77,Max:90.4,Min:81.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.263873
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-2_wildtype-Dpretreated_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 ("McCormac_2-2_wildtype-Dpretreated_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: McCormac_2-2_wildtype-Dpretreated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrients1.5% sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentcontrol wild type seedlings i.e. did not receive far-red before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.255599
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:
RawQ3.69
NoiseAvg:6.12,Stdev:0.33,Max:7.3,Min:5.6
BackgroundAvg:94.39,Stdev:3.16,Max:104.4,Min:87.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.255599
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-3_gun-mutant-Frprecon_Rep1_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 ("McCormac_2-3_gun-mutant-Frprecon_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: McCormac_2-3_gun-mutant-Frprecon_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: gun1,gun5 double mutant
Stock Code:
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentgun1,gun5 double mutant seedlings which were pre-conditioned under far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.260281
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:
RawQ3.68
NoiseAvg:5.91,Stdev:0.22,Max:6.6,Min:5.3
BackgroundAvg:97.91,Stdev:2.04,Max:104.0,Min:94.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.260281
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-4_gun-mutant-Dpretreated_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 ("McCormac_2-4_gun-mutant-Dpretreated_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: McCormac_2-4_gun-mutant-Dpretreated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: gun1,gun5 double mutant
Stock Code:
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentgun1,gun5 double mutant seedlings which did not receive far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.288256
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:
RawQ3.95
NoiseAvg:6.45,Stdev:0.41,Max:7.6,Min:5.4
BackgroundAvg:99.54,Stdev:5.59,Max:113.8,Min:89.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.288256
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-10_phyAmutant-Dpretreated_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 ("McCormac_2-10_phyAmutant-Dpretreated_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: McCormac_2-10_phyAmutant-Dpretreated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: phyA-null mutant
Stock Code: N6219
Growth Conditions:
locationGrowth Room
Temperature22
Lighting(Source: 150)continuous
MediumM&S medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentphyA mutant seedlings which did not receive far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.322864
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:
RawQ3.61
NoiseAvg:5.40,Stdev:0.26,Max:6.2,Min:4.8
BackgroundAvg:87.54,Stdev:3.09,Max:94.6,Min:80.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.322864
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-9_phyAmutant-FRprecon_Rep1_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 ("McCormac_2-9_phyAmutant-FRprecon_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: McCormac_2-9_phyAmutant-FRprecon_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: phyA-null mutant
Stock Code: N6219
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentphyA mutant seedlings which were pre-conditioned under far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.299064
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:
RawQ3.18
NoiseAvg:5.31,Stdev:0.63,Max:7.3,Min:4.2
BackgroundAvg:80.57,Stdev:4.25,Max:92.8,Min:73.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.299064
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Hybridisation Set: McCormac_genome_Exp2_Rep2

Slide: McCormac_2-5_wildtype-Frprecon_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 ("McCormac_2-5_wildtype-Frprecon_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: McCormac_2-5_wildtype-Frprecon_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentwild type seedlings pre-conditioned under far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.273391
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:
RawQ4.57
NoiseAvg:9.53,Stdev:0.53,Max:10.8,Min:8.1
BackgroundAvg:126.15,Stdev:4.36,Max:140.5,Min:119.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.273391
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-6_wildtype-Dpretreated_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 ("McCormac_2-6_wildtype-Dpretreated_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: McCormac_2-6_wildtype-Dpretreated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Treatment5 micromolar Norflurazon
Nutrients1.5% sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentcontrol wild type seedlings i.e. did not receive far-red before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.259357
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:
RawQ4.07
NoiseAvg:7.21,Stdev:0.26,Max:7.8,Min:6.6
BackgroundAvg:108.57,Stdev:5.03,Max:118.2,Min:98.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.259357
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-7_gun-mutant-Frprecon_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 ("McCormac_2-7_gun-mutant-Frprecon_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: McCormac_2-7_gun-mutant-Frprecon_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: gun1,gun5 double mutant
Stock Code:
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentgun1,gun5 double mutant seedlings which were pre-conditioned under far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.265548
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:
RawQ3.94
NoiseAvg:6.51,Stdev:0.41,Max:7.7,Min:5.6
BackgroundAvg:100.74,Stdev:3.98,Max:113.2,Min:92.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.265548
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: McCormac_2-8_gun-mutant-Dpretreated_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 ("McCormac_2-8_gun-mutant-Dpretreated_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: McCormac_2-8_gun-mutant-Dpretreated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: gun1,gun5 double mutant
Stock Code:
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Temperature22
Lighting(Source: 150)continuous
MediumMandS medium
Nutrientsno sucrose
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treatmentgun1,gun5 double mutant seedlings which did not receive far-red light before transfer to white light.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: 2-step LiCl/ Qiagen method
Method:
ProtocolSeedlings were ground in liquid Nitrogen, homogenised in extraction buffer (1:1 mix of acidified phenol: 0.1M LiCl, 0.1M Tris.HCl pH 8.0, 10mM EDTA, 1%w/v SDS), partitioned with chloroform and precipitated with an equal volume 4M LiCl, followed by an ethanol precipitation. The resulting pellet was resuspended and subjected to the Qiagen RNeasy Plant Mini kit. The final phase was partitioned in acid phenol/ chloroform and ethanol precipitated.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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: 2003-07-08

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:0.339484
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:
RawQ3.31
NoiseAvg:5.14,Stdev:0.29,Max:6.1,Min:4.6
BackgroundAvg:88.06,Stdev:3.54,Max:96.8,Min:81.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.339484
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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