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Experiment: Genes Involved in Meristemoid Cell Fate

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-388

The objective of this experiment is to identify genes that are regulated by the LRR-receptor-like protein TOO MANY MOUTHS (TMM) to control stomatal precursor (meristemoid) cell fate and behavior. An interesting aspect of the tmm phenotype is that mutant plants overproduce stomata on leaves, but lack stomata entirely on inflorescence stems and in other locations on the plant. Despite the failure to produce mature stomata, mutant stems produce presumptive stomatal precursors through asymmetric divisions. This suggests that normal TMM signaling is required to maintain meristemoid cell fate but not to execute formative asymmetric divisions. Because stem stomata develop in a morphogenetic wave from tip to base, it is possible to harvest tissues that contain meristemoids but not guard mother cells or mature stomata. This provides an opportunity to compare the gene expression profiles of tissues with meristemoids that maintain their cellular identity and ultimately produce stomata (Col gl1), to tissues in which meristemoids form but rapidly lose their cellular identity due to a failure in TMM signaling (tmm-1). Because guard mother cells and stomata are not present in the tissue harvested, genes that change in abundance during these developmental stages will be filtered out. Ultimately we hope to reveal genes that operate downstream of TMM to confer meristemoid cell fate or to provide 'niche factors' suitable for their development into stomata.

For analysis, approximately 90 developing inflorescence stem tips were collected and flower buds and meristems removed prior to flash freezing for each replicate sample. The region harvested had been previously determined to contain developing meristemoids but not stomata or guard mother cells by microscopic observation. An identical region was harvested from tmm-1 mutant plants grown at the same time, in the same flat.

About the Experimenter

Name: Jeanette Nadeau
Head of Lab Name: Jeanette Nadeau
Lab: Nadeau lab
Institute: University of Central Florida
Address:Department of Biology
4000 Central Florida Blvd.
Orlando, FL
Postcode: 32816-2368
Country: USA
 
Telephone Number: 407-823-1663
Fax Number: 407-823-5769

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:4
 
Experimental Parameters:
gene_knock_out
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Nadeau: Genome set

Slide: Nadeau_1-1_tmm1-inflorescence-tips-pool1_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 ("Nadeau_1-1_tmm1-inflorescence-tips-pool1_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: Nadeau_1-1_tmm1-inflorescence-tips-pool1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: tmm-1
Stock Code: CS6140
Age: 30-40 days
Growth Conditions:
Growth protocolSeeds were planted with toothpicks on the surface of sterilized Promix BX in 4 inch pots placed in covered 1020 flats. Pots were placed in the growth chamber and the covers were removed after one week. Plants were grown at 22°C, 70% humidity and 16 hours light, 125 µmol/m2 sec light intensity. Plants were subirrigated with deionized water once per week, and fertilized with Peter's Fertilizer (1.27g/L) once per week
LocationGrowth chamber
Growth substrateCommercial soil: Promix BX manufactured by Premier Horticulture. Sphagnum, perlite, vermiculite, micro and macronutrients, wetting agent
Developmental Stage:
developmental-stage(Source: Boye's Key [Paradigm Genetics])6.10
Tissue: inflorescence tip
Diseased: Normal
Additional Organism Information:
separation-techniqueThe inflorescence stem containing immature epidermis was harvested as follows: open flowers and buds were rapidly removed from the developing inflorescence stem at the base of the pedicels using a dissecting microscope and fine point forceps. Meristems were then removed from the tip. Tips of the inflorescence stem were removed from the plant and flash frozen in microcentrifuge tubes. Each sample was composed of pooled tips from ~90 inflorescences. Prior to harvesting stems for RNA, approximately 20 inflorescence tips were examined with a microscope to identify the region of the stem that consistently contained developing stomatal meristemoids in the epidermis, but few guard mother cells or mature stomata. These stems were not included in the RNA sample
sampledescriptionBolting Col-3 gl1 plants
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.909374713898
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.09
NoiseAvg:2.87,Stdev:0.09,Max:3.3,Min:2.7
Central-Avg:6507,Count:9
Corner+Avg:121,Count:32
Corner-Avg:8270,Count:32
BackgroundAvg:58.32,Stdev:0.38,Max:59.5,Min:57.4
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.909374713898
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Nadeau_1-2_Col1g11-inflorescence-tips-pool1_Rep1_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Nadeau_1-2_Col1g11-inflorescence-tips-pool1_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: Nadeau_1-2_Col1g11-inflorescence-tips-pool1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-3 gl1
Stock Code:
Age: 30-40 days
Growth Conditions:
Growth protocolSeeds were planted with toothpicks on the surface of sterilized Promix BX in 4 inch pots placed in covered 1020 flats. Pots were placed in the growth chamber and the covers were removed after one week. Plants were grown at 22°C, 70% humidity and 16 hours light, 125 µmol/m2 sec light intensity. Plants were subirrigated with deionized water once per week, and fertilized with Peter's Fertilizer (1.27g/L) once per week
LocationGrowth chamber
Growth substrateCommercial soil: Promix BX manufactured by Premier Horticulture. Sphagnum, perlite, vermiculite, micro and macronutrients, wetting agent
Developmental Stage:
developmental-stage(Source: Boye's Key [Paradigm Genetics])6.10
Tissue: inflorescence tip
Diseased: Normal
Additional Organism Information:
separation-techniqueThe inflorescence stem containing immature epidermis was harvested as follows: open flowers and buds were rapidly removed from the developing inflorescence stem at the base of the pedicels using a dissecting microscope and fine point forceps. Meristems were then removed from the tip. Tips of the inflorescence stem were removed from the plant and flash frozen in microcentrifuge tubes. Each sample was composed of pooled tips from ~90 inflorescences. Prior to harvesting stems for RNA, approximately 20 inflorescence tips were examined with a microscope to identify the region of the stem that consistently contained developing stomatal meristemoids in the epidermis, but few guard mother cells or mature stomata. These stems were not included in the RNA sample
sampledescriptionBolting Col-3 gl1 plants
Other Information: Lehle Seeds

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.963500142097
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.19
NoiseAvg:2.96,Stdev:0.12,Max:3.4,Min:2.7
Central-Avg:5968,Count:9
Corner+Avg:114,Count:32
Corner-Avg:8444,Count:32
BackgroundAvg:61.84,Stdev:0.57,Max:63.1,Min:60.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.963500142097
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Nadeau_1-3_tmm1-inflorescence-tips-pool2_Rep1_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Nadeau_1-3_tmm1-inflorescence-tips-pool2_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: Nadeau_1-3_tmm1-inflorescence-tips-pool2_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: tmm-1
Stock Code: CS6140
Age: 30-40 days
Growth Conditions:
Growth protocolSeeds were planted with toothpicks on the surface of sterilized Promix BX in 4 inch pots placed in covered 1020 flats. Pots were placed in the growth chamber and the covers were removed after one week. Plants were grown at 22°C, 70% humidity and 16 hours light, 125 µmol/m2 sec light intensity. Plants were subirrigated with deionized water once per week, and fertilized with Peter's Fertilizer (1.27g/L) once per week
LocationGrowth chamber
Growth substrateCommercial soil: Promix BX manufactured by Premier Horticulture. Sphagnum, perlite, vermiculite, micro and macronutrients, wetting agent
Developmental Stage:
developmental-stage(Source: Boye's Key [Paradigm Genetics])6.10
Tissue: inflorescence tip
Diseased: Normal
Additional Organism Information:
separation-techniqueThe inflorescence stem containing immature epidermis was harvested as follows: open flowers and buds were rapidly removed from the developing inflorescence stem at the base of the pedicels using a dissecting microscope and fine point forceps. Meristems were then removed from the tip. Tips of the inflorescence stem were removed from the plant and flash frozen in microcentrifuge tubes. Each sample was composed of pooled tips from ~90 inflorescences. Prior to harvesting stems for RNA, approximately 20 inflorescence tips were examined with a microscope to identify the region of the stem that consistently contained developing stomatal meristemoids in the epidermis, but few guard mother cells or mature stomata. These stems were not included in the RNA sample
sampledescriptionBolting Col-3 gl1 plants
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.128508090973
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.01
NoiseAvg:5.57,Stdev:0.25,Max:6.3,Min:5.0
Central-Avg:4078,Count:9
Corner+Avg:169,Count:32
Corner-Avg:6008,Count:32
BackgroundAvg:85.51,Stdev:1.27,Max:88.2,Min:82.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.128508090973
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Nadeau_1-4_Col1g11-inflorescence-tips-pool2_Rep1_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Nadeau_1-4_Col1g11-inflorescence-tips-pool2_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: Nadeau_1-4_Col1g11-inflorescence-tips-pool2_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-3 gl1
Stock Code:
Age: 30-40 days
Growth Conditions:
Growth protocolSeeds were planted with toothpicks on the surface of sterilized Promix BX in 4 inch pots placed in covered 1020 flats. Pots were placed in the growth chamber and the covers were removed after one week. Plants were grown at 22°C, 70% humidity and 16 hours light, 125 µmol/m2 sec light intensity. Plants were subirrigated with deionized water once per week, and fertilized with Peter's Fertilizer (1.27g/L) once per week
LocationGrowth chamber
Growth substrateCommercial soil: Promix BX manufactured by Premier Horticulture. Sphagnum, perlite, vermiculite, micro and macronutrients, wetting agent
Developmental Stage:
developmental-stage(Source: Boye's Key [Paradigm Genetics])6.10
Tissue: inflorescence tip
Diseased: Normal
Additional Organism Information:
separation-techniqueThe inflorescence stem containing immature epidermis was harvested as follows: open flowers and buds were rapidly removed from the developing inflorescence stem at the base of the pedicels using a dissecting microscope and fine point forceps. Meristems were then removed from the tip. Tips of the inflorescence stem were removed from the plant and flash frozen in microcentrifuge tubes. Each sample was composed of pooled tips from ~90 inflorescences. Prior to harvesting stems for RNA, approximately 20 inflorescence tips were examined with a microscope to identify the region of the stem that consistently contained developing stomatal meristemoids in the epidermis, but few guard mother cells or mature stomata. These stems were not included in the RNA sample
sampledescriptionBolting Col-3 gl1 plants
Other Information: Lehle Seeds

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.044398188591
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.13
NoiseAvg:2.60,Stdev:0.08,Max:2.8,Min:2.4
Central-Avg:4441,Count:9
Corner+Avg:77,Count:32
Corner-Avg:5699,Count:32
BackgroundAvg:57.59,Stdev:0.77,Max:59.2,Min:56.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.044398188591
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team