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Experiment: Seedling transcriptome affected by Norflurazon-induced photobleaching of chloroplasts

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-51

Regulation of expression of genes encoding chloroplast components is critical to the autotrophic plant and never more so than in the cotyledons of the de-etiolating seedling. Many chloroplast proteins are nuclear-encoded and a retrograde signal from the chloroplasts (the Plastid Signal) modulates nuclear transcription. However, not all chloroplast-targeted genes are subject to this control and not all plastid-dependent nuclear genes are chloroplast-targeted. We therefore aim to provide the most comprehensive screen yet of which genes are affected by plastid-signalling. To specifically knock-out positive plastid signalling in light-grown cotyledons, the herbicide Norflurazon (NF) is supplied in the growth medium, causing a carotenoid deficiency that leaves the chloroplasts vulnerable to photobleaching. This blocks the expression of a subset of nuclear genes, such as Lhcb and HEMA1.

Two pairs of RNAs will directly compare the transcription in seedlings grown under continuous white light with and without NF. A third RNA will also be compared from a mutant that shows a degree of constitutive positive plastid signalling. These two mutants act synergistically to counteract the effect of NF on nuclear transcription. The gun1,gun5 double mutant maintains a significantly higher level of Lhcb and HEMA1 expression in the presence of photobeached chloroplasts than the NF-treated wild-type. This transcriptome set will therefore complement RNA1 (wild-type+NF) and indicate which of the genes identified from the RNA1/RNA2 comparison are subject to the particular gun1/gun5 plastid signalling pathway(s).

The growth of conditions of the seedlings (namely on MS medium supplemented with 1.5% sucrose, for 3 days under continuous WL following 2 days germination in darkness) has been chosen from the results of our own recent studies using Northern blotting techniques that show these conditions to maximise the respective NF and gun mutant effects on Lhcb and HEMA1 gene expression. This experiment is part one of a two-part study to compare the transcriptional output of this NF-affected pathway with that of a newly discovered FR-mediated pathway. A subsequent array experiment will assess the nuclear response as affected by this FR/ phyA-input pathway and the two sets of array data will be compared and contrasted.

Note:

Col-0 wild-type (NASC code N1092). gun1,gun5 double mutant(obtained from Enriquez Lopez-Juez, Royal Holloway, University of London.(Mochizuki et al. 2001 PNAS 98: 2053-2058). This line cannot be donated by us as it is the IP of Joanne Chory (SALK Institute, USA). Treatment = a herbicide (Norflurazon) application which leads to chloroplast photobleaching and hence down regulation of nuclear genes dependent on plastid signalling from intact chloroplasts.

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:6
 
Experimental Parameters:
parameter gene_knock_out
parametercompound_based_treatment
Quality Control Measures Taken:
no-plants-pooled100 seedlings
References:
 
Other Information:
ArrayExpressAccessionE-NASC-40

Slides in this Experiment

Hybridisation Set: McCormac_genome_Exp1_Rep1

Slide: McCormac_1-1_wildtype-NFtreated_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_1-1_wildtype-NFtreated_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_1-1_wildtype-NFtreated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
Humidityin sealed petri dish therefore 80%
Temperature23oC
Lighting(Source: 250micromol.m-2.s-1 white light)3d growth in darkness followed by 3d continuous white light
MediumMurashige and Skoog basal salts solidified with agar
Treatmentthe herbicide Norflurazon was included in the medium at 5 microMolar
Nutrients1.5 w/v sucrose
Stratificationno (seeds were vernalised prior to germination)
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: whole young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentWT seedlings treated with the herbicide Norflurazon.
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

Production Date: 2002-11-12
Date Hybridised: 2002-11-18

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:0.352
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

Slide: McCormac_1-2_wildtype-Contrl_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_1-2_wildtype-Contrl_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_1-2_wildtype-Contrl_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
Humidityin sealed petri dish therefore 80%
Temperature23oC
Lighting(Source: 250micromol.m-2.s-1 white light)3d growth in darkness followed by 3d continuous white light
MediumMurashige and Skoog basal salts solidified with agar
Treatmentcontrol
Nutrients1.5 w/v sucrose
Stratificationno (seeds were vernalised prior to germination)
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: whole young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentWT seedlings which had not been treated with Norflurazon and therefore are the control sample.
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

Production Date: 2002-11-12
Date Hybridised: 2002-11-18

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:0.586
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

Slide: McCormac_1-3_mutant-NFtreated_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_1-3_mutant-NFtreated_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_1-3_mutant-NFtreated_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: gun1,gun5 double mutant
Stock Code:
Growth Conditions:
locationGrowth Room
Humidityin sealed petri dish therefore 80%
Temperature23oC
Lighting(Source: 250micromol.m-2.s-1 white light)3d growth in darkness followed by 3d continuous white light
MediumMurashige and Skoog basal salts solidified with agar
Treatmentgrowth medium supplemented with 5 micromolar Norflurazon
Nutrients1.5 w/v sucrose
Stratificationno (seeds were vernalised prior to germination)
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: whole young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentSeedlings of a double mutant line gun1,gun5 which had been treated with Norflurazon.
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

Production Date: 2002-11-12
Date Hybridised: 2002-11-18

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:0.484
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

Hybridisation Set: McCormac_genome_Exp1_Rep2

Slide: McCormac_1-4_wildtype-NFtreated_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_1-4_wildtype-NFtreated_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_1-4_wildtype-NFtreated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidityin sealed petri dish therefore 80%
Temperature23oC
Lighting(Source: 250micromol.m-2.s-1 white light)3d growth in darkness followed by 3d continuous white light
MediumMurashige and Skoog basal salts solidified with agar
Treatment5 micromolar Norflurazon
Nutrients1.5 w/v sucrose
Stratificationno (seeds were vernalised prior to germination)
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: whole young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentSeedlings of WT which had been treated with Norflurazon.
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

Production Date: 2002-11-12
Date Hybridised: 2002-11-18

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:0.472
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

Slide: McCormac_1-5_wildtype-Contrl_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_1-5_wildtype-Contrl_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_1-5_wildtype-Contrl_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code: N1092
Growth Conditions:
locationGrowth Room
AtmosphereNormal
Humidityin sealed petri dish therefore 80%
Temperature23oC
Lighting(Source: 250micromol.m-2.s-1 white light)3d growth in darkness followed by 3d continuous white light
MediumMurashige and Skoog basal salts solidified with agar
Treatmentcontrol
Nutrients1.5 w/v sucrose
Stratificationno (seeds were vernalised prior to germination)
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: whole young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentSeedlings of WT which had not been treated with Norflurazon.
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

Production Date: 2002-11-12
Date Hybridised: 2002-11-18

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:0.512
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

Slide: McCormac_1-6_mutant-NFtreated_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_1-6_mutant-NFtreated_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_1-6_mutant-NFtreated_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: gun1,gun5 double mutant
Stock Code:
Growth Conditions:
locationGrowth Room
Humidityin sealed petri dish therefore 80%
Temperature23oC
Lighting(Source: 250micromol.m-2.s-1 white light)3d growth in darkness followed by 3d continuous white light
MediumMurashige and Skoog basal salts solidified with agar
Treatmentgrowth medium supplemented with 5 micromolar Norflurazon
Nutrients1.5 w/v sucrose
Stratificationno (seeds were vernalised prior to germination)
Developmental Stage:
developm-stage(Source: Boyes et al., Plant Cell 2001 13, 1499)1.0-cotyledons_fully_opened
Tissue: whole young seedlings in entirety with cotyledons, hypocotyl and primary roots - (no emerged true leaves).
Diseased: Normal
in vivo Treatment:
treamentSeedlings of a double mutant line gun1,gun5 treated with Norflurazon.
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

Production Date: 2002-11-12
Date Hybridised: 2002-11-18

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:0.514
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


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