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Experiment: Molecular responses to external pH changes in roots of Arabidopsis thaliana

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-470

Plants live in soils that vary considerably, both spatially and over time, in terms of nutrient composition and pH. Consistently, plants have to recognize and adapt to these changes by altering their structure and metabolism. The goal of this array analysis is to characterize the global transcriptional response to external pH changes in roots, which to date is almost unexplored. Arabidopsis thaliana (Columbia-0) were grown in hydroponic cultures in basic nutrient solution. Two days before treatment the media was shifted to nutrient solution containing 5mM MES, pH 6. At the time of the treatment start (4 hours after light on) the plants were shifted to nutrient solutions of pH 4.5 and 6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.

About the Experimenter

Name:Dr Ida Lager
Head of Lab Name:Prof Allan Rasmusson
Lab:
Address:Department of Cell and Organism Biology
Lund University
Soelvegatan 35
Lund
Postcode: 22326
Country: Sweden
 

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: growth_condition_design
Number of Slides:12
 
Experimental Parameters:
parameterchange_pH
Quality Control Measures Taken:
no-plants-pooled
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Lager: Molecular responses to external pH changes in roots of Arabidopsis thaliana_genome

Slide: Lager_1-1_1hr-pH4.5_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 ("Lager_1-1_1hr-pH4.5_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: Lager_1-1_1hr-pH4.5_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.803482115269
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.99
NoiseAvg:2.10,Stdev:0.05,Max:2.2,Min:2.0
Central-Avg:6239,Count:9
Corner+Avg:57,Count:32
Corner-Avg:7074,Count:32
BackgroundAvg:46.05,Stdev:0.33,Max:46.8,Min:45.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.803482115269
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-2_1hr-pH4.5_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Lager_1-2_1hr-pH4.5_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: Lager_1-2_1hr-pH4.5_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.951458513737
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:
RawQ2.14
NoiseAvg:2.10,Stdev:0.06,Max:2.3,Min:1.9
Central-Avg:5807,Count:9
Corner+Avg:63,Count:32
Corner-Avg:6709,Count:32
BackgroundAvg:49.87,Stdev:0.33,Max:50.7,Min:48.6
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.951458513737
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-3_1hr-pH4.5_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Lager_1-3_1hr-pH4.5_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lager_1-3_1hr-pH4.5_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.497754126787
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:
RawQ2.98
NoiseAvg:3.35,Stdev:0.09,Max:3.8,Min:3.2
Central-Avg:6554,Count:9
Corner+Avg:79,Count:32
Corner-Avg:7098,Count:32
BackgroundAvg:76.22,Stdev:0.61,Max:77.5,Min:74.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.497754126787
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-4_1hr-pH6_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 ("Lager_1-4_1hr-pH6_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: Lager_1-4_1hr-pH6_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.72625619173
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:
RawQ2.42
NoiseAvg:2.63,Stdev:0.07,Max:2.9,Min:2.5
Central-Avg:4933,Count:9
Corner+Avg:70,Count:32
Corner-Avg:6334,Count:32
BackgroundAvg:58.95,Stdev:0.37,Max:59.6,Min:57.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.726256191730
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-5_1hr-pH6_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 ("Lager_1-5_1hr-pH6_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: Lager_1-5_1hr-pH6_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.512749791145
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:
RawQ2.83
NoiseAvg:3.41,Stdev:0.11,Max:3.7,Min:3.2
Central-Avg:7008,Count:9
Corner+Avg:80,Count:32
Corner-Avg:8482,Count:32
BackgroundAvg:73.31,Stdev:0.81,Max:74.7,Min:69.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.512749791145
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-6_1hr-pH6_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Lager_1-6_1hr-pH6_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lager_1-6_1hr-pH6_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.640706300735
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:
RawQ2.71
NoiseAvg:3.14,Stdev:0.12,Max:3.4,Min:2.9
Central-Avg:6338,Count:9
Corner+Avg:77,Count:32
Corner-Avg:8153,Count:32
BackgroundAvg:68.68,Stdev:0.53,Max:69.9,Min:67.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.640706300735
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-7_8hr-pH4.5_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 ("Lager_1-7_8hr-pH4.5_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: Lager_1-7_8hr-pH4.5_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.490563511848
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:
RawQ2.42
NoiseAvg:2.80,Stdev:0.06,Max:3.0,Min:2.6
Central-Avg:8393,Count:9
Corner+Avg:85,Count:32
Corner-Avg:9438,Count:32
BackgroundAvg:58.47,Stdev:0.49,Max:59.4,Min:57.0
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.490563511848
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-8_8hr-pH4.5_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 ("Lager_1-8_8hr-pH4.5_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: Lager_1-8_8hr-pH4.5_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.698720395565
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.24
NoiseAvg:2.39,Stdev:0.06,Max:2.6,Min:2.3
Central-Avg:8345,Count:9
Corner+Avg:84,Count:32
Corner-Avg:8774,Count:32
BackgroundAvg:54.87,Stdev:0.23,Max:55.7,Min:54.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.698720395565
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-9_8hr-pH4.5_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Lager_1-9_8hr-pH4.5_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lager_1-9_8hr-pH4.5_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.955479383469
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:
RawQ2.00
NoiseAvg:2.07,Stdev:0.06,Max:2.3,Min:2.0
Central-Avg:6035,Count:9
Corner+Avg:62,Count:32
Corner-Avg:7326,Count:32
BackgroundAvg:50.24,Stdev:0.43,Max:51.2,Min:48.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.955479383469
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-10_8hr-pH6_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 ("Lager_1-10_8hr-pH6_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: Lager_1-10_8hr-pH6_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.565735042095
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:
RawQ2.37
NoiseAvg:2.57,Stdev:0.14,Max:3.3,Min:2.3
Central-Avg:5567,Count:9
Corner+Avg:64,Count:32
Corner-Avg:6845,Count:32
BackgroundAvg:58.21,Stdev:0.58,Max:59.5,Min:55.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.565735042095
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-11_8hr-pH6_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 ("Lager_1-11_8hr-pH6_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: Lager_1-11_8hr-pH6_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.954927563667
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:2.07,Stdev:0.07,Max:2.3,Min:1.8
Central-Avg:6518,Count:9
Corner+Avg:62,Count:32
Corner-Avg:6818,Count:32
BackgroundAvg:52.43,Stdev:0.68,Max:53.7,Min:50.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.954927563667
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Lager_1-12_8hr-pH6_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Lager_1-12_8hr-pH6_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Lager_1-12_8hr-pH6_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0 (Columbia)
Stock Code: N1092
Growth Conditions:
Stratificationtwo days at 4C
Sterilisation1 min 70% EtOH 5 min 50% bleach, 0.5% Tween-20 Rinse with water 3 times
ProtocolSeeds were germinated in tubes filled with 0.7% agarose, tubes, tubes were transferred to hydroponic cultures after germination.The hydroponic cultures were not kept sterile. The media used is a basic nutrient solution as previously described (Somerville and Ogren, 1982) but at half strength [2.5 mM KNO3, 1.25 mM KH2PO4 (pH5.6), 1 mM MgSO4, 1 mM Ca(NO3)2, 25 microM Fe- EDTA and the reported micronutrient mix at 1x concentration.
Temperature23 °C average, 23 °C day, 23 °C night
Humiditynot controlled % average, not controlled % day, not controlled % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)3.90
Tissue: roots
in vivo Treatment: Two days before treatment the growth media was shifted to new media (Somerville media, same as described in growth) including 5mM MES, pH was set to 6.0. Treatment was started 4 hours after light on. The growth media was shifted to new Somerville media with 5mM MES at pH4.5 or pH6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
Additional Organism Information:
Sample DescriptionHydroponically grown Arabidopsis thaliana (Col-0). Plants were used before bolting occured.
Other Information:
Timecourse start procedureswitch of media

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:0.970098674297
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:
RawQ2.77
NoiseAvg:2.86,Stdev:0.07,Max:3.0,Min:2.6
Central-Avg:5521,Count:9
Corner+Avg:58,Count:32
Corner-Avg:6244,Count:32
BackgroundAvg:67.12,Stdev:1.32,Max:70.0,Min:63.3
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.970098674297
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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