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Experiment: An investigation into transcriptional changes in the developing Arabidopsis leaf caused by a novel signalling protein, SPH1. (BBSRC grant 6/P18254)

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-65

Arabidopsis genome sequencing has revealed the presence of at least three extensive gene families that may encode protein ligands. One of these, the SPH (S-protein homologue) family, was identified as a direct result of our studies on self-incompatibility in Papaver. The Arabidopsis SPH gene family consists of 81 members. We have initiated experimental work on a subset of these. RT-PCR studies indicate that many, if not all, SPH genes are expressed. Each SPH gene encodes an N-terminal signal peptide sequence and thus SPH proteins are likely to be secreted. Until recently none of the genes in this family had known function. However we have evidence that one member of the family, SPH1 is involved in leaf vascular development. In order to determine the function of SPH1, Arabidopsis plants were transformed with an SPH1 antisense construct. Analysis of the mutant phenotype shows that whilst plants appear as wt until principal growth stage 1.04, they subsequently show severe morphological defects. Plants are severely dwarfed with twisted rosette leaves at ~ 30% wt length and width as well as shortened inflorescence stem. Closer examination revealed aberrant leaf vasculature and severe reduction in expansion of parenchyma cells surrounding the primary leaf vein. We have conducted preliminary immunolocalisation studies with antibody raised to SPH1. These suggest that SPH1 protein is secreted by cells within the developing vasculature of the immature leaf (leaves<6mm in length). The data that we have so far obtained leads us to believe that SPH1 protein acts as a signalling molecule during early leaf development. As SPH1 is likely to be a signalling protein it is assumed that its interaction with a cognate receptor results in initiation of developmental processes within leaf tissue. The purpose of the experiment is to determine the network of genes within the normally developing rosette leaf whose expression is altered by SPH1. This will be accomplished by comparing transcriptional levels in rosette leaves of antisense-SPH1 plants with wt plants. We propose to make target RNA from rosette leaves taken from plants at principal stage 1.05 (immediately after initial appearance of developmental abnormality) and from plants at principal stage 1.14 (where gross changes are apparent). We propose to use replicate slides for each hybridisation, i.e. 2 hybridised to RNA from wt leaves at 1.05, 2 antisense at 1.05, 2 wt at 1.14 and 2 antisense at 1.14.

About the Experimenter

Name:Dr Mike Wheeler
Head of Lab Name:Prof Chris Franklin
Lab:
Institute: University of Birmingham
Address:School of Biosciences
University of Birmingham
Edgbaston
Birmingham
Postcode: B15 2TT
Country: UK
 
Telephone Number: 0121 414 5913

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:12
 
Experimental Parameters:
Parameterinduced_mutation
Quality Control Measures Taken:
no-plants-pooled40
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Wheeler_genome

Slide: Wheeler-w05_SLD_Rep1

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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 ("Wheeler-w05_SRC_Rep1") 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: Wheeler-w05_SRC_Rep1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-09

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:
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: Wheeler-w05_SLD_Rep2

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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 ("Wheeler-w05_SRC_Rep2") 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: Wheeler-w05_SRC_Rep2
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-09

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:
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: Wheeler-a05_SLD_Rep1

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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 ("Wheeler-a05_SRC_Rep1") 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: Wheeler-a05_SRC_Rep1
Organism: Arabidopsis thaliana
Alias: SPH1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: RNAi construct in SPH1
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-09

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:
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: Wheeler-a5_SLD_Rep2

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 ("Wheeler-a05_SRC_Rep2") 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: Wheeler-a05_SRC_Rep2
Organism: Arabidopsis thaliana
Alias: SPH1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: RNAi construct in SPH1
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-09

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:
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: Wheeler-w14_SLD_Rep1

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 ("Wheeler-w14_SRC_Rep1") 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: Wheeler-w14_SRC_Rep1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.14
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-31

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:
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: Wheeler-w14_SLD_Rep2

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 ("Wheeler-w14_SRC_Rep2") 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: Wheeler-w14_SRC_Rep2
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.14
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-31

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:
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: Wheeler-a14_SLD_Rep1

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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 ("Wheeler-a14_SRC_Rep1") 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: Wheeler-a14_SRC_Rep1
Organism: Arabidopsis thaliana
Alias: SPH1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.14
Genetic Variation: RNAi construct in SPH1
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-31

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:
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: Wheeler-a14_SLD_Rep2

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 ("Wheeler-a14_SRC_Rep2") 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: Wheeler-a14_SRC_Rep2
Organism: Arabidopsis thaliana
Alias: SPH1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.14
Genetic Variation: RNAi construct in SPH1
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-08-06

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:
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: Wheeler-a05_SLD_Rep3

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 ("Wheeler-a05_SRC_Rep3") 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: Wheeler-a05_SRC_Rep3
Organism: Arabidopsis thaliana
Alias: SPH1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: RNAi construct in SPH1
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-09

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:0.46
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: Wheeler-a14_SLD_Rep3

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 ("Wheeler-a14_SRC_Rep3") 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: Wheeler-a14_SRC_Rep3
Organism: Arabidopsis thaliana
Alias: SPH1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.14
Genetic Variation: RNAi construct in SPH1
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-08-06

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:
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: Wheeler-w5_SLD_Rep3

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 ("Wheeler-w05_SRC_Rep3") 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: Wheeler-w05_SRC_Rep3
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-09

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:
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: Wheeler-w14_SLD_Rep3

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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 ("Wheeler-w14_SRC_Rep3") 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: Wheeler-w14_SRC_Rep3
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
locationGreenhouse
AtmosphereNormal
Humidity80%
Temperature20C
Lighting(Source: 10 000 lux)16h photoperiod
Mediumcompost
TreatmentIntersept
NutrientsJohn Innes compost and perlite
Waterdaily from below
Stratification4 days at 4C
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.14
Tissue: rosette leaves
Diseased: Normal
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Wheeler Trizol Protocol
Method:
Protocol500mg tissue ground in liquid nitrogen with mortar and pestle transferred to 50ml Falcon tube and 15ml Trizol added -to 60C for 10min Centrifuged at 12000g for 10 min at 4C, SN transferred to fresh 50ml tube. 3ml chloroform added, vortex 15s. RT for 2 min. Centrifuged at 10000g 15min at 4C. Aq. phase collected and placed in new 50ml tube. RNA precipitated using 0.5 vol isopropanol and 0.5 vol 0.8M Na citate/1.2M NaCl. Mixed and set at Rt for 10 min. Centrifuged at 10,000g for 10 min at 4C. SN discarded, pellet washed with 20ml 75% ethanol. Vortexed 10s. Centrifuged at 10000g, 10 min at 4C. Pellet dried in air for 10 min. 300 microlitres of DEPC-water added + 1 microlitre of RNase Inhibitor. Concentration checked by spec and by gel electrophoresis. Cleaned up using QIAgen RNA minikits. Frozen on dry ice.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol

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: 2003-07-31

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:
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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