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Experiment: Arabidopsis pvip mutants

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-395

Transcriptional comparison of Arabidopsis thaliana pvip1 ; pvip2 RNAi double mutants against Col-0 wildtype;Tissue used: whole rosettes of 4-week old plants grown under short-day conditions (8h light; 20oC); 3 biological replicates for each, control = Col-0 and mutant = pvip1 pvip2;PVIP = Potyvirus-VPg-interacting protein (Dunoyer et al. 2004 J. Virol. 78: 2301-2309).

About the Experimenter

Name:Dr Dominik Schmidt
Head of Lab Name:Prof Andy Maule
Lab:
Institute: John Innes Centre
Address:Disease and Stress Biology
John Innes Centre
Colney
Norwich
Postcode: NR4 7UH
Country: United Kingdom
 
Telephone Number: 01603 450214
Fax Number: 01603 450045

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: strain_or_line_design;
Number of Slides:6
 
Experimental Parameters:
parameterstrain_or_line
Quality Control Measures Taken:
References:
referenceDunoyer et al. 2004 J. Virol. 78: 2301-2309
 
Other Information:

Slides in this Experiment

Hybridisation Set: Schmidt: Arabidopsis pvip mutants_genome

Slide: Schmidt_DS-C1_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 ("Schmidt_DS-C1_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: Schmidt_DS-C1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
stratificationAfter sowing on plates seeds were stored in the dark at 4°C for 3 days
sterilisationDry surface-sterilisation with chlorine gas generated by 100 ml household bleach and 3 ml concentrated HCl
Growth protocolSeeds of Arabidopsis thaliana ecotype Columbia (Col-0) and PVIP1 and PVIP2 RNAi transgenic lines (pvip1/pvip2; Dunoyer et al 2004, J Virol 78: 2301) were surface-sterilised in a desiccator by chlorine gas generated with a mixture of 100 ml commercial bleach and 3 ml concentrated hydrochloric acid for 4 h. Seeds were sown on MS agar (0.8%) that contained 20 mg/l phosphinothricin (Basta) for the pvip1/pvip2 line. After stratification in the dark at 4°C for 3 days the plates with the seeds were transferred to a growth room (20°C, 16 h light). 14 days after germination Col-0 plants and Basta-resistant pvip1/pvip2 plants were transferred to soil (Scotts Levington Special Custom Mix Compost with Intercept 5GR) and grown under short-day conditions (20°C, 8 h light, 70% relative air humidity) for 3 weeks
LocationGrowth Room
growth substrate typeCommercial soil: Scotts Levington Special Custom Mix Compost with Intercept 5GR
Average temperture20°C
Average humidity70%
Developmental Stage: 35 days (1.13- Boyes Key)
Tissue: whole rosettes
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:1.635958
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:
RawQ2.31
NoiseAvg:2.98,Stdev:0.10,Max:3.3,Min:2.6
BackgroundAvg:65.97,Stdev:0.25,Max:66.5,Min:65.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.635958
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Schmidt_DS-C2_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Schmidt_DS-C2_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: Schmidt_DS-C2_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
stratificationAfter sowing on plates seeds were stored in the dark at 4°C for 3 days
sterilisationDry surface-sterilisation with chlorine gas generated by 100 ml household bleach and 3 ml concentrated HCl
Growth protocolSeeds of Arabidopsis thaliana ecotype Columbia (Col-0) and PVIP1 and PVIP2 RNAi transgenic lines (pvip1/pvip2; Dunoyer et al 2004, J Virol 78: 2301) were surface-sterilised in a desiccator by chlorine gas generated with a mixture of 100 ml commercial bleach and 3 ml concentrated hydrochloric acid for 4 h. Seeds were sown on MS agar (0.8%) that contained 20 mg/l phosphinothricin (Basta) for the pvip1/pvip2 line. After stratification in the dark at 4°C for 3 days the plates with the seeds were transferred to a growth room (20°C, 16 h light). 14 days after germination Col-0 plants and Basta-resistant pvip1/pvip2 plants were transferred to soil (Scotts Levington Special Custom Mix Compost with Intercept 5GR) and grown under short-day conditions (20°C, 8 h light, 70% relative air humidity) for 3 weeks
LocationGrowth Room
growth substrate typeCommercial soil: Scotts Levington Special Custom Mix Compost with Intercept 5GR
Average temperture20°C
Average humidity70%
Developmental Stage: 35 days (1.13- Boyes Key)
Tissue: whole rosettes
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:1.421926
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:
RawQ2.11
NoiseAvg:2.66,Stdev:0.16,Max:3.4,Min:2.3
BackgroundAvg:56.47,Stdev:0.54,Max:57.8,Min:55.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.421926
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Schmidt_DS-C3_Re3p_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 ("Schmidt_DS-C3_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: Schmidt_DS-C3_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Growth Conditions:
stratificationAfter sowing on plates seeds were stored in the dark at 4°C for 3 days
sterilisationDry surface-sterilisation with chlorine gas generated by 100 ml household bleach and 3 ml concentrated HCl
Growth protocolSeeds of Arabidopsis thaliana ecotype Columbia (Col-0) and PVIP1 and PVIP2 RNAi transgenic lines (pvip1/pvip2; Dunoyer et al 2004, J Virol 78: 2301) were surface-sterilised in a desiccator by chlorine gas generated with a mixture of 100 ml commercial bleach and 3 ml concentrated hydrochloric acid for 4 h. Seeds were sown on MS agar (0.8%) that contained 20 mg/l phosphinothricin (Basta) for the pvip1/pvip2 line. After stratification in the dark at 4°C for 3 days the plates with the seeds were transferred to a growth room (20°C, 16 h light). 14 days after germination Col-0 plants and Basta-resistant pvip1/pvip2 plants were transferred to soil (Scotts Levington Special Custom Mix Compost with Intercept 5GR) and grown under short-day conditions (20°C, 8 h light, 70% relative air humidity) for 3 weeks
LocationGrowth Room
growth substrate typeCommercial soil: Scotts Levington Special Custom Mix Compost with Intercept 5GR
Average temperture20°C
Average humidity70%
Developmental Stage: 35 days (1.13- Boyes Key)
Tissue: whole rosettes
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:1.674909
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:
RawQ2.57
NoiseAvg:3.13,Stdev:0.10,Max:3.6,Min:2.8
BackgroundAvg:73.21,Stdev:0.81,Max:75.4,Min:71.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.674909
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Schmidt_DS-M1_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 ("Schmidt_DS-M1_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: Schmidt_DS-M1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pvip1/pvip2
Stock Code:
Genetic Background: Col-3
Growth Conditions:
stratificationAfter sowing on plates seeds were stored in the dark at 4°C for 3 days.
sterilisationDry surface-sterilisation with chlorine gas generated by 100 ml household bleach and 3 ml concentrated HCl.
Growth protocolSeeds of Arabidopsis thaliana ecotype Columbia (Col-0) and PVIP1 and PVIP2 RNAi transgenic lines (pvip1/pvip2; Dunoyer et al 2004, J Virol 78: 2301) were surface-sterilised in a desiccator by chlorine gas generated with a mixture of 100 ml commercial bleach and 3 ml concentrated hydrochloric acid for 4 h. Seeds were sown on MS agar (0.8%) that contained 20 mg/l phosphinothricin (Basta) for the pvip1/pvip2 line. After stratification in the dark at 4°C for 3 days the plates with the seeds were transferred to a growth room (20°C, 16 h light). 14 days after germination Col-0 plants and Basta-resistant pvip1/pvip2 plants were transferred to soil (Scotts Levington Special Custom Mix Compost with Intercept 5GR) and grown under short-day conditions (20°C, 8 h light, 70% relative air humidity) for 3 weeks.
LocationGrowth Room
growth substrate typeCommercial soil: Scotts Levington Special Custom Mix Compost with Intercept 5GR
Average temperture20°C
Average humidity70%
Developmental Stage: 35 days (1.13- Boyes Key)
Genetic Variation: RNAi: gene knock out
Tissue: whole rosettes
Additional Organism Information: Genes knocked out: At5g48160 and At3g07780
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:0.970089
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:3.05,Stdev:0.10,Max:3.4,Min:2.8
BackgroundAvg:60.84,Stdev:0.61,Max:62.6,Min:59.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.970089
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Schmidt_DS-M2_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 ("Schmidt_DS-M2_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: Schmidt_DS-M2_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pvip1/pvip2
Stock Code:
Genetic Background: Col-3
Growth Conditions:
stratificationAfter sowing on plates seeds were stored in the dark at 4°C for 3 days.
sterilisationDry surface-sterilisation with chlorine gas generated by 100 ml household bleach and 3 ml concentrated HCl.
Growth protocolSeeds of Arabidopsis thaliana ecotype Columbia (Col-0) and PVIP1 and PVIP2 RNAi transgenic lines (pvip1/pvip2; Dunoyer et al 2004, J Virol 78: 2301) were surface-sterilised in a desiccator by chlorine gas generated with a mixture of 100 ml commercial bleach and 3 ml concentrated hydrochloric acid for 4 h. Seeds were sown on MS agar (0.8%) that contained 20 mg/l phosphinothricin (Basta) for the pvip1/pvip2 line. After stratification in the dark at 4°C for 3 days the plates with the seeds were transferred to a growth room (20°C, 16 h light). 14 days after germination Col-0 plants and Basta-resistant pvip1/pvip2 plants were transferred to soil (Scotts Levington Special Custom Mix Compost with Intercept 5GR) and grown under short-day conditions (20°C, 8 h light, 70% relative air humidity) for 3 weeks.
LocationGrowth Room
growth substrate typeCommercial soil: Scotts Levington Special Custom Mix Compost with Intercept 5GR
Average temperture20°C
Average humidity70%
Developmental Stage: 35 days (1.13- Boyes Key)
Genetic Variation: RNAi: gene knock out
Tissue: whole rosettes
Additional Organism Information: Genes knocked out: At5g48160 and At3g07780
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:1.238107
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:
RawQ2.10
NoiseAvg:2.88,Stdev:0.22,Max:4.2,Min:2.6
BackgroundAvg:56.49,Stdev:0.51,Max:58.2,Min:55.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.238107
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Schmidt_DS-M3_Rep3_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 ("Schmidt_DS-M3_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: Schmidt_DS-M3_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pvip1/pvip2
Stock Code:
Genetic Background: Col-3
Growth Conditions:
stratificationAfter sowing on plates seeds were stored in the dark at 4°C for 3 days.
sterilisationDry surface-sterilisation with chlorine gas generated by 100 ml household bleach and 3 ml concentrated HCl.
Growth protocolSeeds of Arabidopsis thaliana ecotype Columbia (Col-0) and PVIP1 and PVIP2 RNAi transgenic lines (pvip1/pvip2; Dunoyer et al 2004, J Virol 78: 2301) were surface-sterilised in a desiccator by chlorine gas generated with a mixture of 100 ml commercial bleach and 3 ml concentrated hydrochloric acid for 4 h. Seeds were sown on MS agar (0.8%) that contained 20 mg/l phosphinothricin (Basta) for the pvip1/pvip2 line. After stratification in the dark at 4°C for 3 days the plates with the seeds were transferred to a growth room (20°C, 16 h light). 14 days after germination Col-0 plants and Basta-resistant pvip1/pvip2 plants were transferred to soil (Scotts Levington Special Custom Mix Compost with Intercept 5GR) and grown under short-day conditions (20°C, 8 h light, 70% relative air humidity) for 3 weeks.
LocationGrowth Room
growth substrate typeCommercial soil: Scotts Levington Special Custom Mix Compost with Intercept 5GR
Average temperture20°C
Average humidity70%
Developmental Stage: 35 days (1.13- Boyes Key)
Genetic Variation: RNAi: gene knock out
Tissue: whole rosettes
Additional Organism Information: Genes knocked out: At5g48160 and At3g07780
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:1.289793
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:
RawQ2.05
NoiseAvg:3.13,Stdev:0.69,Max:6.4,Min:2.5
BackgroundAvg:56.07,Stdev:0.49,Max:57.7,Min:54.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.289793
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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