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Experiment: The effects of infection with wild-type or mutant cucumber mosaic virus on the Arabidopsis transcriptome

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-552

The cucumber mosaic virus (CMV) 2b counter-defense protein disrupts plant antiviral mechanisms mediated by RNA silencing and salicylic acid (SA). Using NASC ATH-1 microarrays to investigate defensive gene expression in 2b-transgenic Arabidopsis thaliana plants we found that, surprisingly, 2b inhibited expression of few SA-regulated genes and in some instances enhanced the effect of SA on certain genes. Strikingly, the 2b protein inhibited changes in the expression of 90% of genes regulated by jasmonic acid (NASCARRAYS-415: Lewsey et al. Molec. Plant-Microbe Interact. vol. 23, in press). We will extend this work to understand the effects of the 2b protein on plant gene expression during an authentic viral infection. By comparing the effects on the transcriptome of infection by wild-type CMV and the 2b gene deletion mutant, CMV∆2b, we will reveal not only the influence of the 2b protein but also the effects of its interactions with other viral gene products (and the process of infection itself), on the host transcriptome.

About the Experimenter

Name:Dr Zhiyou Du
Head of Lab Name: John Carr
Lab:
Address:Department of Plant Sciences, University of Cambridge
Downing street, Cambridge, Cambridgeshire, CB2 3EA, UNITED KINGDOM
Postcode:
Country:
 
Telephone Number: 01223330215

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: pathogenicity_design
Number of Slides:9
 
Experimental Parameters:
parameterinfect
Quality Control Measures Taken:
References:
 
Other Information:
other factorsvirus challenge

Slides in this Experiment

Hybridisation Set: Du: The effects of infection with wild-type or mutant cucumber mosaic virus on the Arabidopsis transcriptome

Slide: Du_552-1_Mock-At_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 ("Du_552-1_Mock-At_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: Du_552-1_Mock-At_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with distilled water when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.785843
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.215906
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.967468
Spike_AFFX-r2-Bs-dap_5_signal102.003281
NoiseAvg:1.98,Std:0.07,Min:1.9,Max:2.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal42.670120
#P14435
Spike_AFFX-r2-Bs-phe_M_signal45.268349
Corner-Avg:8815,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal149.945221
Spike_AFFX-r2-Ec-bioB_3_signal116.959030
Spike_AFFX-r2-Bs-lys_M_signal19.072271
Spike_AFFX-r2-P1-cre_3_signal7493.251953
Spike_AFFX-r2-Bs-lys_3-5-ratio1.782774
Spike_AFFX-r2-Bs-dap_M_signal231.053696
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.178837
Spike_AFFX-r2-Ec-bioB_avg-signal129.265396
Spike_AFFX-r2-Bs-thr_avg-signal74.026695
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1351.625366
Spike_AFFX-r2-Bs-phe_5_signal37.798725
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2281.498047
RawQ1.971243
Spike_AFFX-r2-Bs-lys_5_signal29.047176
Signal(A)3.980756
%A35.028496
Signal(All)154.439545
Corner+Avg:112,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal571.648438
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal56.031040
Spike_AFFX-r2-Ec-bioD_avg-signal1816.561768
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.283646
Spike_AFFX-r2-Bs-lys_avg-signal33.301331
Spike_AFFX-r2-P1-cre_avg-signal6827.970703
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.687856
Spike_AFFX-r2-Bs-thr_3_signal92.971222
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal46.366039
Spike_AFFX-r2-Bs-dap_3_signal300.497864
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.687966
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal520.108948
#M385
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal51.784546
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.099094
Spike_AFFX-r2-Bs-thr_M_signal86.438751
Signal(P)241.396500
Spike_AFFX-r2-Bs-phe_3-5-ratio1.482353
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:6034,Count:9
Spike_AFFX-r2-P1-cre_5_signal6162.688965
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7990
Signal(M)16.626505
BackgroundAvg:47.65,Std:0.40,Min:46.5,Max:48.7
Spike_AFFX-r2-Ec-bioC_avg-signal545.878662
Spike_AFFX-r2-Bs-dap_avg-signal211.184952
Spike_AFFX-r2-Bs-dap_3-5-ratio2.945963
Spike_AFFX-r2-Ec-bioB_5_signal120.891930
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.785843
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-2_CMV-WT_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 ("Du_552-2_CMV-WT_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: Du_552-2_CMV-WT_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with wildtype Fny-CMV (CMV-WT) when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.935059
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.092831
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.850290
Spike_AFFX-r2-Bs-dap_5_signal95.095688
NoiseAvg:1.90,Std:0.06,Min:1.7,Max:2.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal38.305962
#P14487
Spike_AFFX-r2-Bs-phe_M_signal32.560684
Corner-Avg:7893,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal155.274887
Spike_AFFX-r2-Ec-bioB_3_signal107.170876
Spike_AFFX-r2-Bs-lys_M_signal18.789837
Spike_AFFX-r2-P1-cre_3_signal6702.391113
Spike_AFFX-r2-Bs-lys_3-5-ratio1.978574
Spike_AFFX-r2-Bs-dap_M_signal195.898239
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.740989
Spike_AFFX-r2-Ec-bioB_avg-signal129.495377
Spike_AFFX-r2-Bs-thr_avg-signal73.095528
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1353.021729
Spike_AFFX-r2-Bs-phe_5_signal42.879986
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2244.384766
RawQ1.915687
Spike_AFFX-r2-Bs-lys_5_signal17.831507
Signal(A)4.099358
%A34.945198
Signal(All)149.667297
Corner+Avg:109,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal505.429993
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal30.493237
Spike_AFFX-r2-Ec-bioD_avg-signal1798.703247
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.511620
Spike_AFFX-r2-Bs-lys_avg-signal23.967436
Spike_AFFX-r2-P1-cre_avg-signal6417.721191
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.543183
Spike_AFFX-r2-Bs-thr_3_signal104.996231
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal35.311302
Spike_AFFX-r2-Bs-dap_3_signal277.238342
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.658794
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal512.187378
#M352
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal35.280960
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.986807
Spike_AFFX-r2-Bs-thr_M_signal75.984383
Signal(P)232.992035
Spike_AFFX-r2-Bs-phe_3-5-ratio0.711130
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:4664,Count:9
Spike_AFFX-r2-P1-cre_5_signal6133.051270
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7971
Signal(M)16.703487
BackgroundAvg:48.82,Std:0.37,Min:47.5,Max:49.7
Spike_AFFX-r2-Ec-bioC_avg-signal508.808685
Spike_AFFX-r2-Bs-dap_avg-signal189.410767
Spike_AFFX-r2-Bs-dap_3-5-ratio2.915362
Spike_AFFX-r2-Ec-bioB_5_signal126.040337
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.935059
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-3_CMV-A2b_mutant_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 ("Du_552-3_CMV-A2b_mutant_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: Du_552-3_CMV-A2b_mutant_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with purified Fny-CMV∆2b (CMV-∆2b) virion of 100 ng/µl when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:1.064359
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.242908
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.786227
Spike_AFFX-r2-Bs-dap_5_signal108.338066
NoiseAvg:1.85,Std:0.02,Min:1.8,Max:1.9
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal41.939873
#P13978
Spike_AFFX-r2-Bs-phe_M_signal26.266033
Corner-Avg:7843,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal162.034088
Spike_AFFX-r2-Ec-bioB_3_signal108.779213
Spike_AFFX-r2-Bs-lys_M_signal15.447718
Spike_AFFX-r2-P1-cre_3_signal8541.295898
Spike_AFFX-r2-Bs-lys_3-5-ratio2.409312
Spike_AFFX-r2-Bs-dap_M_signal218.604172
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.393091
Spike_AFFX-r2-Ec-bioB_avg-signal136.389771
Spike_AFFX-r2-Bs-thr_avg-signal70.036865
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1337.956055
Spike_AFFX-r2-Bs-phe_5_signal34.603081
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2036.827026
RawQ1.923827
Spike_AFFX-r2-Bs-lys_5_signal24.118664
Signal(A)5.018255
%A36.711967
Signal(All)155.924698
Corner+Avg:105,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal705.541321
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal46.155567
Spike_AFFX-r2-Ec-bioD_avg-signal1687.391602
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.280140
Spike_AFFX-r2-Bs-lys_avg-signal32.558582
Spike_AFFX-r2-P1-cre_avg-signal7706.660156
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.007891
Spike_AFFX-r2-Bs-thr_3_signal100.365944
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal35.674892
Spike_AFFX-r2-Bs-dap_3_signal257.467560
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.522342
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal581.559448
#M458
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal58.109375
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.213189
Spike_AFFX-r2-Bs-thr_M_signal67.804787
Signal(P)250.791565
Spike_AFFX-r2-Bs-phe_3-5-ratio1.333857
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5286,Count:9
Spike_AFFX-r2-P1-cre_5_signal6872.024414
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8374
Signal(M)19.770491
BackgroundAvg:48.17,Std:0.25,Min:47.5,Max:48.8
Spike_AFFX-r2-Ec-bioC_avg-signal643.550415
Spike_AFFX-r2-Bs-dap_avg-signal194.803268
Spike_AFFX-r2-Bs-dap_3-5-ratio2.376520
Spike_AFFX-r2-Ec-bioB_5_signal138.356018
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.064359
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-4_Mock-At_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 ("Du_552-4_Mock-At_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: Du_552-4_Mock-At_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with distilled water when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.911546
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.074263
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.059518
Spike_AFFX-r2-Bs-dap_5_signal94.579201
NoiseAvg:1.91,Std:0.05,Min:1.7,Max:2.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal33.930248
#P14276
Spike_AFFX-r2-Bs-phe_M_signal26.461758
Corner-Avg:7891,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal157.172119
Spike_AFFX-r2-Ec-bioB_3_signal121.256813
Spike_AFFX-r2-Bs-lys_M_signal15.795647
Spike_AFFX-r2-P1-cre_3_signal6908.128418
Spike_AFFX-r2-Bs-lys_3-5-ratio1.198789
Spike_AFFX-r2-Bs-dap_M_signal171.087204
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.315623
Spike_AFFX-r2-Ec-bioB_avg-signal130.958054
Spike_AFFX-r2-Bs-thr_avg-signal57.273014
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1205.960571
Spike_AFFX-r2-Bs-phe_5_signal28.603300
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2002.601074
RawQ1.900949
Spike_AFFX-r2-Bs-lys_5_signal20.551462
Signal(A)4.377357
%A35.756248
Signal(All)154.478958
Corner+Avg:106,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal576.677429
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal31.968988
Spike_AFFX-r2-Ec-bioD_avg-signal1604.280762
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.586586
Spike_AFFX-r2-Bs-lys_avg-signal20.327990
Spike_AFFX-r2-P1-cre_avg-signal6669.352539
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.657168
Spike_AFFX-r2-Bs-thr_3_signal78.569672
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal29.011351
Spike_AFFX-r2-Bs-dap_3_signal215.072876
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.660586
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal551.150513
#M378
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal24.636866
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.046316
Spike_AFFX-r2-Bs-thr_M_signal59.319130
Signal(P)243.873322
Spike_AFFX-r2-Bs-phe_3-5-ratio1.117668
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5987,Count:9
Spike_AFFX-r2-P1-cre_5_signal6430.576172
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8156
Signal(M)17.004648
BackgroundAvg:48.23,Std:0.36,Min:47.2,Max:49.2
Spike_AFFX-r2-Ec-bioC_avg-signal563.913940
Spike_AFFX-r2-Bs-dap_avg-signal160.246429
Spike_AFFX-r2-Bs-dap_3-5-ratio2.273998
Spike_AFFX-r2-Ec-bioB_5_signal114.445236
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.911546
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-5_CMV-WT_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 ("Du_552-5_CMV-WT_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: Du_552-5_CMV-WT_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with wildtype Fny-CMV (CMV-WT) when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:1.072041
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.211721
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.800570
Spike_AFFX-r2-Bs-dap_5_signal112.802505
NoiseAvg:1.85,Std:0.05,Min:1.7,Max:2.0
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal35.868095
#P14118
Spike_AFFX-r2-Bs-phe_M_signal30.088131
Corner-Avg:7950,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal159.424881
Spike_AFFX-r2-Ec-bioB_3_signal113.806435
Spike_AFFX-r2-Bs-lys_M_signal14.193750
Spike_AFFX-r2-P1-cre_3_signal8241.828125
Spike_AFFX-r2-Bs-lys_3-5-ratio1.507214
Spike_AFFX-r2-Bs-dap_M_signal203.188354
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.403467
Spike_AFFX-r2-Ec-bioB_avg-signal138.462692
Spike_AFFX-r2-Bs-thr_avg-signal67.037193
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1254.473633
Spike_AFFX-r2-Bs-phe_5_signal52.179874
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2258.520996
RawQ1.978487
Spike_AFFX-r2-Bs-lys_5_signal27.954214
Signal(A)4.951479
%A36.321789
Signal(All)154.273804
Corner+Avg:102,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal579.796204
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal36.482601
Spike_AFFX-r2-Ec-bioD_avg-signal1756.497314
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.893906
Spike_AFFX-r2-Bs-lys_avg-signal28.093649
Spike_AFFX-r2-P1-cre_avg-signal7521.791016
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.784305
Spike_AFFX-r2-Bs-thr_3_signal86.207794
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal39.583538
Spike_AFFX-r2-Bs-dap_3_signal305.942963
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.800373
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal510.074036
#M407
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal42.132977
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.136690
Spike_AFFX-r2-Bs-thr_M_signal79.035675
Signal(P)245.833511
Spike_AFFX-r2-Bs-phe_3-5-ratio0.699170
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5628,Count:9
Spike_AFFX-r2-P1-cre_5_signal6801.753906
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8285
Signal(M)17.898987
BackgroundAvg:47.69,Std:0.38,Min:47.0,Max:48.8
Spike_AFFX-r2-Ec-bioC_avg-signal544.935120
Spike_AFFX-r2-Bs-dap_avg-signal207.311279
Spike_AFFX-r2-Bs-dap_3-5-ratio2.712200
Spike_AFFX-r2-Ec-bioB_5_signal142.156754
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.072041
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-6_CMV-A2b_mutant_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 ("Du_552-6_CMV-A2b_mutant_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: Du_552-6_CMV-A2b_mutant_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with purified Fny-CMVΔ2b (CMV-Δ2b) virion of 100 ng/µl when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.898901
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.242415
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.944960
Spike_AFFX-r2-Bs-dap_5_signal118.077156
NoiseAvg:1.93,Std:0.06,Min:1.8,Max:2.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal42.630562
#P13756
Spike_AFFX-r2-Bs-phe_M_signal39.282536
Corner-Avg:8618,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal153.474167
Spike_AFFX-r2-Ec-bioB_3_signal109.211685
Spike_AFFX-r2-Bs-lys_M_signal18.513481
Spike_AFFX-r2-P1-cre_3_signal7923.025879
Spike_AFFX-r2-Bs-lys_3-5-ratio1.526470
Spike_AFFX-r2-Bs-dap_M_signal273.943695
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.247833
Spike_AFFX-r2-Ec-bioB_avg-signal126.086205
Spike_AFFX-r2-Bs-thr_avg-signal93.579704
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1405.968994
Spike_AFFX-r2-Bs-phe_5_signal53.430134
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2302.766602
RawQ2.029473
Spike_AFFX-r2-Bs-lys_5_signal31.053436
Signal(A)4.840915
%A37.597546
Signal(All)158.186996
Corner+Avg:119,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal567.783020
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal65.584534
Spike_AFFX-r2-Ec-bioD_avg-signal1854.367798
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.306885
Spike_AFFX-r2-Bs-lys_avg-signal32.323013
Spike_AFFX-r2-P1-cre_avg-signal7150.070313
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.095572
Spike_AFFX-r2-Bs-thr_3_signal138.456955
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal52.765732
Spike_AFFX-r2-Bs-dap_3_signal385.914520
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.637850
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal518.301208
#M478
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal47.402126
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.095469
Spike_AFFX-r2-Bs-thr_M_signal99.651588
Signal(P)258.673920
Spike_AFFX-r2-Bs-phe_3-5-ratio1.227482
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5579,Count:9
Spike_AFFX-r2-P1-cre_5_signal6377.115234
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8576
Signal(M)17.597414
BackgroundAvg:48.29,Std:0.29,Min:47.6,Max:49.0
Spike_AFFX-r2-Ec-bioC_avg-signal543.042114
Spike_AFFX-r2-Bs-dap_avg-signal259.311798
Spike_AFFX-r2-Bs-dap_3-5-ratio3.268325
Spike_AFFX-r2-Ec-bioB_5_signal115.572754
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.898901
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-7_Mock-At_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 ("Du_552-7_Mock-At_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: Du_552-7_Mock-At_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with distilled water when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.787853
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.203764
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.992931
Spike_AFFX-r2-Bs-dap_5_signal204.818146
NoiseAvg:1.84,Std:0.06,Min:1.7,Max:2.0
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal71.518654
#P14354
Spike_AFFX-r2-Bs-phe_M_signal72.673820
Corner-Avg:8342,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal179.060684
Spike_AFFX-r2-Ec-bioB_3_signal150.979538
Spike_AFFX-r2-Bs-lys_M_signal31.294407
Spike_AFFX-r2-P1-cre_3_signal8191.008789
Spike_AFFX-r2-Bs-lys_3-5-ratio1.225484
Spike_AFFX-r2-Bs-dap_M_signal461.975647
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.660464
Spike_AFFX-r2-Ec-bioB_avg-signal160.698227
Spike_AFFX-r2-Bs-thr_avg-signal137.552734
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1560.193848
Spike_AFFX-r2-Bs-phe_5_signal79.292992
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2309.691895
RawQ1.865909
Spike_AFFX-r2-Bs-lys_5_signal49.547588
Signal(A)3.804934
%A35.256466
Signal(All)156.027359
Corner+Avg:110,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal641.485352
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal77.785912
Spike_AFFX-r2-Ec-bioD_avg-signal1934.942871
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.928539
Spike_AFFX-r2-Bs-lys_avg-signal47.187252
Spike_AFFX-r2-P1-cre_avg-signal7497.753906
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.814993
Spike_AFFX-r2-Bs-thr_3_signal190.272812
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal76.584236
Spike_AFFX-r2-Bs-dap_3_signal585.822815
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.480388
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal589.827087
#M414
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal60.719757
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.087582
Spike_AFFX-r2-Bs-thr_M_signal150.866745
Signal(P)245.355301
Spike_AFFX-r2-Bs-phe_3-5-ratio0.980994
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5823,Count:9
Spike_AFFX-r2-P1-cre_5_signal6804.498535
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8042
Signal(M)15.833004
BackgroundAvg:46.42,Std:0.32,Min:45.6,Max:47.4
Spike_AFFX-r2-Ec-bioC_avg-signal615.656250
Spike_AFFX-r2-Bs-dap_avg-signal417.538849
Spike_AFFX-r2-Bs-dap_3-5-ratio2.860209
Spike_AFFX-r2-Ec-bioB_5_signal152.054474
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.787853
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-8_CMV-WT_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 ("Du_552-8_CMV-WT_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: Du_552-8_CMV-WT_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with purified wildtype Fny-CMV (CMV-WT) virion of 100 ng/µl when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 16 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.732217
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.238732
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.895911
Spike_AFFX-r2-Bs-dap_5_signal131.837189
NoiseAvg:2.00,Std:0.04,Min:1.9,Max:2.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal43.222271
#P14436
Spike_AFFX-r2-Bs-phe_M_signal48.660030
Corner-Avg:8250,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal130.816116
Spike_AFFX-r2-Ec-bioB_3_signal102.189842
Spike_AFFX-r2-Bs-lys_M_signal21.702780
Spike_AFFX-r2-P1-cre_3_signal7143.908691
Spike_AFFX-r2-Bs-lys_3-5-ratio1.451467
Spike_AFFX-r2-Bs-dap_M_signal303.045410
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.591763
Spike_AFFX-r2-Ec-bioB_avg-signal115.689491
Spike_AFFX-r2-Bs-thr_avg-signal96.003601
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1286.247681
Spike_AFFX-r2-Bs-phe_5_signal58.344749
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1957.604492
RawQ2.063410
Spike_AFFX-r2-Bs-lys_5_signal30.767017
Signal(A)3.671151
%A35.129330
Signal(All)150.006012
Corner+Avg:111,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal526.513611
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal52.732368
Spike_AFFX-r2-Ec-bioD_avg-signal1621.926025
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.288033
Spike_AFFX-r2-Bs-lys_avg-signal32.375702
Spike_AFFX-r2-P1-cre_avg-signal6455.510742
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.582639
Spike_AFFX-r2-Bs-thr_3_signal155.244171
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal53.245716
Spike_AFFX-r2-Bs-dap_3_signal404.491760
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.521950
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal470.982635
#M361
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal44.657314
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.117905
Spike_AFFX-r2-Bs-thr_M_signal89.544380
Signal(P)234.620132
Spike_AFFX-r2-Bs-phe_3-5-ratio0.903807
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5765,Count:9
Spike_AFFX-r2-P1-cre_5_signal5767.112305
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8013
Signal(M)14.526626
BackgroundAvg:52.13,Std:0.35,Min:51.4,Max:53.1
Spike_AFFX-r2-Ec-bioC_avg-signal498.748108
Spike_AFFX-r2-Bs-dap_avg-signal279.791473
Spike_AFFX-r2-Bs-dap_3-5-ratio3.068116
Spike_AFFX-r2-Ec-bioB_5_signal114.062538
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.732217
NF1.000000
HZ4
Tau0.015000

Slide: Du_552-9_CMV-A2b_mutant_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 ("Du_552-9_CMV-A2b_mutant_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: Du_552-9_CMV-A2b_mutant_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 5-weeks old
Growth Conditions:
StratificationFour days in dark at 4 celsius degree
ProtocolPlants were grown on 4:1 soil/sand mixture, amended with 0.002% (w/v) intercept 5GR. They were maintained at 21 degrees celsius in a custom built growth room, under cycles of 8 hours light/16 hours dark and at 60% relative humidity.
Soil ConstituentsLevington M3 compost (Scotts, Chilworth, Ipswich, UK)
waterWatered daily until soil was moist
Temperature21 °C average, 21 °C day, 21 °C night
Humidity60 % average, 60 % day, 60 % night
Medium
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)
Tissue: Aerial tissues
in vivo Treatment: Approximately 24 plants were inoculated with purified Fny-CMVΔ2b (CMV-Δ2b) virion of 100 ng/µl when plants were 3 weeks old (growth stage 1.05 to 1.07). Aerial tissues of total 11 plants were harvested 14 days post inoculation ((growth stage 3.50 to 3.70) pooled to minimise variation. The pooled tissue was then used for total RNA extraction.
Additional Organism Information:
Sample Description
Other Information:
ecotype_habitat

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:0.958097
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.198784
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.968636
Spike_AFFX-r2-Bs-dap_5_signal113.872589
NoiseAvg:1.75,Std:0.05,Min:1.6,Max:1.9
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal49.183037
#P14094
Spike_AFFX-r2-Bs-phe_M_signal37.072311
Corner-Avg:7161,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal176.429581
Spike_AFFX-r2-Ec-bioB_3_signal124.045090
Spike_AFFX-r2-Bs-lys_M_signal16.072096
Spike_AFFX-r2-P1-cre_3_signal9059.195313
Spike_AFFX-r2-Bs-lys_3-5-ratio1.393986
Spike_AFFX-r2-Bs-dap_M_signal255.502594
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.754882
Spike_AFFX-r2-Ec-bioB_avg-signal142.845444
Spike_AFFX-r2-Bs-thr_avg-signal88.371338
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1623.826660
Spike_AFFX-r2-Bs-phe_5_signal66.387016
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2269.464111
RawQ1.870833
Spike_AFFX-r2-Bs-lys_5_signal28.978977
Signal(A)4.101946
%A36.448925
Signal(All)158.608032
Corner+Avg:93,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal648.272156
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal53.348049
Spike_AFFX-r2-Ec-bioD_avg-signal1946.645386
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.788689
Spike_AFFX-r2-Bs-lys_avg-signal28.482452
Spike_AFFX-r2-P1-cre_avg-signal8308.090820
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.762385
Spike_AFFX-r2-Bs-thr_3_signal135.493439
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal52.269123
Spike_AFFX-r2-Bs-dap_3_signal316.203461
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.397602
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal538.374634
#M402
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal40.396278
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.204128
Spike_AFFX-r2-Bs-thr_M_signal80.437546
Signal(P)253.799957
Spike_AFFX-r2-Bs-phe_3-5-ratio0.803592
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5258,Count:9
Spike_AFFX-r2-P1-cre_5_signal7556.986328
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8314
Signal(M)16.639362
BackgroundAvg:47.38,Std:0.48,Min:46.2,Max:48.6
Spike_AFFX-r2-Ec-bioC_avg-signal593.323364
Spike_AFFX-r2-Bs-dap_avg-signal228.526199
Spike_AFFX-r2-Bs-dap_3-5-ratio2.776818
Spike_AFFX-r2-Ec-bioB_5_signal128.061630
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.958097
NF1.000000
HZ4
Tau0.015000


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