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Experiment: pCS19

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-545

Identification of genes regulated by ATHB4. Seedlings grown for 7 days under continuous white light of the pCS19.5 line were treated for 4 h with or without a combination of DEX and/or CHX. Reference: Sorin et al. (2009). Plant J 59, 266, 277.

About the Experimenter

Name:Dr Jaime F Martinez-Garcia
Head of Lab Name:Dr Jaime F Martinez-Garcia
Lab:
Address:Department of Molecular Genetics, Centre de Recerca en Agrigenomica (CRAG) - CSIC
Jordi Girona 18, Barcelona, , 08034, SPAIN
Postcode:
Country:
 
Telephone Number: +34-934006189
Fax Number: +34-932045904

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: compound_based_treatment; genetic_modification_design
Number of Slides:12
 
Experimental Parameters:
parameter compound_based_treatment
parametergene_knock_out
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Martinez-Garcia: pCS19

Slide: Martinez-Garcia_545-1_7-day_pCS19.5_4h_ -CHX -DEX_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 ("Martinez-Garcia_545-1_7-day_pCS19.5_4h_ -CHX -DEX_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: Martinez-Garcia_545-1_7-day_pCS19.5_4h_ -CHX -DEX_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 0uM DEX and/or 0uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 0uM DEX and 0uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.916427
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-ratio1.396134
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.925059
Spike_AFFX-r2-Bs-dap_5_signal105.245888
NoiseAvg:2.21,Std:0.05,Min:2.0,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_signal72.634094
#P14619
Spike_AFFX-r2-Bs-phe_M_signal36.704929
Corner-Avg:5963,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal134.450928
Spike_AFFX-r2-Ec-bioB_3_signal102.415932
Spike_AFFX-r2-Bs-lys_M_signal22.478874
Spike_AFFX-r2-P1-cre_3_signal6983.994141
Spike_AFFX-r2-Bs-lys_3-5-ratio1.488894
Spike_AFFX-r2-Bs-dap_M_signal188.974182
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio1.638899
Spike_AFFX-r2-Ec-bioB_avg-signal115.859924
Spike_AFFX-r2-Bs-thr_avg-signal98.735222
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1357.299072
Spike_AFFX-r2-Bs-phe_5_signal45.320446
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1795.825684
RawQ2.253582
Spike_AFFX-r2-Bs-lys_5_signal24.236650
Signal(A)5.268627
%A34.037704
Signal(All)149.433685
Corner+Avg:68,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal563.220581
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal47.598518
Spike_AFFX-r2-Ec-bioD_avg-signal1576.562378
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P64.090309
Spike_AFFX-r2-Bs-lys_avg-signal27.600441
Spike_AFFX-r2-P1-cre_avg-signal5993.187012
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.871986
Spike_AFFX-r2-Bs-thr_3_signal119.039917
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-signal43.207966
Spike_AFFX-r2-Bs-dap_3_signal224.938400
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.323088
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal436.122955
#M427
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal36.085796
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.291426
Spike_AFFX-r2-Bs-thr_M_signal104.531639
Signal(P)229.822754
Spike_AFFX-r2-Bs-phe_3-5-ratio1.050266
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5006,Count:9
Spike_AFFX-r2-P1-cre_5_signal5002.379883
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7764
Signal(M)18.496244
BackgroundAvg:51.98,Std:0.30,Min:51.3,Max:52.9
Spike_AFFX-r2-Ec-bioC_avg-signal499.671753
Spike_AFFX-r2-Bs-dap_avg-signal173.052841
Spike_AFFX-r2-Bs-dap_3-5-ratio2.137265
Spike_AFFX-r2-Ec-bioB_5_signal110.712906
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.916427
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-2_7-day_pCS19.5_4hr_-CHX -DEX_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 ("Martinez-Garcia_545-2_7-day_pCS19.5_4hr_-CHX -DEX_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: Martinez-Garcia_545-2_7-day_pCS19.5_4hr_-CHX -DEX_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 0uM DEX and/or 0uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 0uM DEX and 0uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:1.194394
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-ratio1.218591
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.072040
Spike_AFFX-r2-Bs-dap_5_signal104.895248
NoiseAvg:2.16,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_signal63.316528
#P14065
Spike_AFFX-r2-Bs-phe_M_signal33.186111
Corner-Avg:5007,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal127.831795
Spike_AFFX-r2-Ec-bioB_3_signal100.379234
Spike_AFFX-r2-Bs-lys_M_signal23.906590
Spike_AFFX-r2-P1-cre_3_signal5967.125000
Spike_AFFX-r2-Bs-lys_3-5-ratio2.055596
Spike_AFFX-r2-Bs-dap_M_signal230.795731
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.973472
Spike_AFFX-r2-Ec-bioB_avg-signal107.281624
Spike_AFFX-r2-Bs-thr_avg-signal119.785744
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1221.523804
Spike_AFFX-r2-Bs-phe_5_signal68.532753
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1750.742920
RawQ2.173399
Spike_AFFX-r2-Bs-lys_5_signal26.637955
Signal(A)6.491790
%A36.128891
Signal(All)151.052124
Corner+Avg:63,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal486.009338
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal53.289753
Spike_AFFX-r2-Ec-bioD_avg-signal1486.133301
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.661552
Spike_AFFX-r2-Bs-lys_avg-signal35.100475
Spike_AFFX-r2-P1-cre_avg-signal5431.932617
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.209557
Spike_AFFX-r2-Bs-thr_3_signal188.269897
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-signal51.669537
Spike_AFFX-r2-Bs-dap_3_signal248.033676
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.433245
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal490.402222
#M504
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal54.756882
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.991042
Spike_AFFX-r2-Bs-thr_M_signal107.770813
Signal(P)240.361816
Spike_AFFX-r2-Bs-phe_3-5-ratio0.777581
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:3857,Count:9
Spike_AFFX-r2-P1-cre_5_signal4896.739746
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8241
Signal(M)22.442766
BackgroundAvg:53.05,Std:0.53,Min:52.0,Max:54.5
Spike_AFFX-r2-Ec-bioC_avg-signal488.205780
Spike_AFFX-r2-Bs-dap_avg-signal194.574890
Spike_AFFX-r2-Bs-dap_3-5-ratio2.364584
Spike_AFFX-r2-Ec-bioB_5_signal93.633842
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.194394
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-3_7-day_pCS19.5_4hr_-CHX -DEX_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 ("Martinez-Garcia_545-3_7-day_pCS19.5_4hr_-CHX -DEX_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: Martinez-Garcia_545-3_7-day_pCS19.5_4hr_-CHX -DEX_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 0uM DEX and/or 0uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 0uM DEX and 0uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.78765
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-ratio1.295175
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.064252
Spike_AFFX-r2-Bs-dap_5_signal146.466507
NoiseAvg:2.25,Std:0.07,Min:2.0,Max:2.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal59.319901
#P14849
Spike_AFFX-r2-Bs-phe_M_signal65.348907
Corner-Avg:8851,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal158.260468
Spike_AFFX-r2-Ec-bioB_3_signal127.225395
Spike_AFFX-r2-Bs-lys_M_signal20.008718
Spike_AFFX-r2-P1-cre_3_signal7152.314453
Spike_AFFX-r2-Bs-lys_3-5-ratio1.121818
Spike_AFFX-r2-Bs-dap_M_signal268.908325
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.613551
Spike_AFFX-r2-Ec-bioB_avg-signal135.010117
Spike_AFFX-r2-Bs-thr_avg-signal107.815308
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1441.959106
Spike_AFFX-r2-Bs-phe_5_signal64.160736
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2103.438965
RawQ2.220964
Spike_AFFX-r2-Bs-lys_5_signal32.167973
Signal(A)5.064176
%A32.981148
Signal(All)150.514603
Corner+Avg:102,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal471.254791
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal55.517765
Spike_AFFX-r2-Ec-bioD_avg-signal1772.698975
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P65.098640
Spike_AFFX-r2-Bs-lys_avg-signal29.421097
Spike_AFFX-r2-P1-cre_avg-signal6337.295410
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.920210
Spike_AFFX-r2-Bs-thr_3_signal155.035568
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-signal61.675800
Spike_AFFX-r2-Bs-dap_3_signal300.355835
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.458737
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal449.948090
#M438
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal36.086597
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.047354
Spike_AFFX-r2-Bs-thr_M_signal109.090485
Signal(P)228.133698
Spike_AFFX-r2-Bs-phe_3-5-ratio0.865292
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:6522,Count:9
Spike_AFFX-r2-P1-cre_5_signal5522.276367
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7523
Signal(M)17.312803
BackgroundAvg:54.02,Std:0.16,Min:53.7,Max:54.7
Spike_AFFX-r2-Ec-bioC_avg-signal460.601440
Spike_AFFX-r2-Bs-dap_avg-signal238.576889
Spike_AFFX-r2-Bs-dap_3-5-ratio2.050679
Spike_AFFX-r2-Ec-bioB_5_signal119.544464
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.787650
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-4_7-day_pCS19.5_4hr_-CHX +DEX_Rep1_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Martinez-Garcia_545-4_7-day_pCS19.5_4hr_-CHX +DEX_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: Martinez-Garcia_545-4_7-day_pCS19.5_4hr_-CHX +DEX_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 5uM DEX and 0uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 5uM DEX and 0uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.946837
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-ratio1.292421
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.111675
Spike_AFFX-r2-Bs-dap_5_signal151.084396
NoiseAvg:2.25,Std:0.06,Min:2.1,Max:2.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal60.662582
#P14527
Spike_AFFX-r2-Bs-phe_M_signal70.436050
Corner-Avg:6530,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal132.052994
Spike_AFFX-r2-Ec-bioB_3_signal114.277298
Spike_AFFX-r2-Bs-lys_M_signal20.247581
Spike_AFFX-r2-P1-cre_3_signal6866.872559
Spike_AFFX-r2-Bs-lys_3-5-ratio1.772426
Spike_AFFX-r2-Bs-dap_M_signal243.323013
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.632592
Spike_AFFX-r2-Ec-bioB_avg-signal116.375893
Spike_AFFX-r2-Bs-thr_avg-signal122.060997
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1300.492798
Spike_AFFX-r2-Bs-phe_5_signal70.618874
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1909.753540
RawQ2.133902
Spike_AFFX-r2-Bs-lys_5_signal31.689463
Signal(A)5.893064
%A34.397194
Signal(All)150.594803
Corner+Avg:72,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal524.550415
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal52.451923
Spike_AFFX-r2-Ec-bioD_avg-signal1605.123169
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.686981
Spike_AFFX-r2-Bs-lys_avg-signal36.034752
Spike_AFFX-r2-P1-cre_avg-signal6090.029297
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.915826
Spike_AFFX-r2-Bs-thr_3_signal159.699829
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-signal64.502281
Spike_AFFX-r2-Bs-dap_3_signal285.410675
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.468485
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal463.588440
#M437
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal56.167213
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.131500
Spike_AFFX-r2-Bs-thr_M_signal145.820587
Signal(P)232.694427
Spike_AFFX-r2-Bs-phe_3-5-ratio0.742747
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5884,Count:9
Spike_AFFX-r2-P1-cre_5_signal5313.185547
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7846
Signal(M)19.401442
BackgroundAvg:54.34,Std:0.44,Min:53.3,Max:55.3
Spike_AFFX-r2-Ec-bioC_avg-signal494.069427
Spike_AFFX-r2-Bs-dap_avg-signal226.606033
Spike_AFFX-r2-Bs-dap_3-5-ratio1.889081
Spike_AFFX-r2-Ec-bioB_5_signal102.797394
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.946837
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-5_7-day_pCS19.5_4hr_-CHX +DEX_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 ("Martinez-Garcia_545-5_7-day_pCS19.5_4hr_-CHX +DEX_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: Martinez-Garcia_545-5_7-day_pCS19.5_4hr_-CHX +DEX_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 5uM DEX and 0uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 5uM DEX and 0uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.839563
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-ratio1.059749
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.008309
Spike_AFFX-r2-Bs-dap_5_signal138.583893
NoiseAvg:2.24,Std:0.07,Min:2.0,Max:2.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal73.134293
#P14587
Spike_AFFX-r2-Bs-phe_M_signal64.003067
Corner-Avg:7121,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal120.676727
Spike_AFFX-r2-Ec-bioB_3_signal94.436745
Spike_AFFX-r2-Bs-lys_M_signal22.267389
Spike_AFFX-r2-P1-cre_3_signal5291.048828
Spike_AFFX-r2-Bs-lys_3-5-ratio1.070749
Spike_AFFX-r2-Bs-dap_M_signal240.650101
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.046217
Spike_AFFX-r2-Ec-bioB_avg-signal102.924004
Spike_AFFX-r2-Bs-thr_avg-signal157.217087
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1083.034790
Spike_AFFX-r2-Bs-phe_5_signal62.415245
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1870.883301
RawQ2.256270
Spike_AFFX-r2-Bs-lys_5_signal52.352913
Signal(A)5.212251
%A34.195530
Signal(All)148.805817
Corner+Avg:87,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal425.035492
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal59.185925
Spike_AFFX-r2-Ec-bioD_avg-signal1476.958984
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.950024
Spike_AFFX-r2-Bs-lys_avg-signal43.559052
Spike_AFFX-r2-P1-cre_avg-signal5141.894043
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.854450
Spike_AFFX-r2-Bs-thr_3_signal222.782913
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-signal61.868084
Spike_AFFX-r2-Bs-dap_3_signal340.617371
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.727445
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal421.383575
#M423
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal56.056850
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.008667
Spike_AFFX-r2-Bs-thr_M_signal175.734070
Signal(P)229.391479
Spike_AFFX-r2-Bs-phe_3-5-ratio0.948261
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:4683,Count:9
Spike_AFFX-r2-P1-cre_5_signal4992.739258
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7800
Signal(M)17.663237
BackgroundAvg:78.68,Std:0.45,Min:77.7,Max:80.4
Spike_AFFX-r2-Ec-bioC_avg-signal423.209534
Spike_AFFX-r2-Bs-dap_avg-signal239.950439
Spike_AFFX-r2-Bs-dap_3-5-ratio2.457842
Spike_AFFX-r2-Ec-bioB_5_signal93.658546
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.839563
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-6_7-day_pCS19.5_4hr_-CHX +DEX_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 ("Martinez-Garcia_545-6_7-day_pCS19.5_4hr_-CHX +DEX_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: Martinez-Garcia_545-6_7-day_pCS19.5_4hr_-CHX +DEX_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 5uM DEX and 0uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 5uM DEX and 0uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.8022
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-ratio1.225891
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.134203
Spike_AFFX-r2-Bs-dap_5_signal104.077171
NoiseAvg:2.10,Std:0.05,Min:2.0,Max:2.2
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal49.419415
#P14893
Spike_AFFX-r2-Bs-phe_M_signal40.058964
Corner-Avg:7138,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal130.585510
Spike_AFFX-r2-Ec-bioB_3_signal121.882530
Spike_AFFX-r2-Bs-lys_M_signal21.869287
Spike_AFFX-r2-P1-cre_3_signal6439.495117
Spike_AFFX-r2-Bs-lys_3-5-ratio1.614416
Spike_AFFX-r2-Bs-dap_M_signal201.338608
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.466443
Spike_AFFX-r2-Ec-bioB_avg-signal119.976341
Spike_AFFX-r2-Bs-thr_avg-signal87.391907
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1115.350464
Spike_AFFX-r2-Bs-phe_5_signal68.528732
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1884.255493
RawQ2.034011
Spike_AFFX-r2-Bs-lys_5_signal24.771822
Signal(A)4.735192
%A33.020603
Signal(All)149.424728
Corner+Avg:84,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal482.131958
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal49.963615
Spike_AFFX-r2-Ec-bioD_avg-signal1499.802979
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P65.291542
Spike_AFFX-r2-Bs-lys_avg-signal28.877710
Spike_AFFX-r2-P1-cre_avg-signal5846.202148
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_signal121.890144
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-signal52.850437
Spike_AFFX-r2-Bs-dap_3_signal231.690521
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.689384
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal449.520020
#M385
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal39.992027
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.072548
Spike_AFFX-r2-Bs-thr_M_signal90.866173
Signal(P)225.981247
Spike_AFFX-r2-Bs-phe_3-5-ratio0.729090
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5141,Count:9
Spike_AFFX-r2-P1-cre_5_signal5252.909180
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7532
Signal(M)18.633495
BackgroundAvg:75.66,Std:0.28,Min:75.0,Max:76.5
Spike_AFFX-r2-Ec-bioC_avg-signal465.825989
Spike_AFFX-r2-Bs-dap_avg-signal179.035416
Spike_AFFX-r2-Bs-dap_3-5-ratio2.226142
Spike_AFFX-r2-Ec-bioB_5_signal107.460983
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.802200
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-7_7-day_pCS19.5_4hr_+CHX -DEX_Rep1_ATH1

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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 ("Martinez-Garcia_545-7_7-day_pCS19.5_4hr_+CHX -DEX_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: Martinez-Garcia_545-7_7-day_pCS19.5_4hr_+CHX -DEX_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 0uM DEX and 50uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 0uM DEX and 50uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.738347
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-ratio1.283180
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.321748
Spike_AFFX-r2-Bs-dap_5_signal90.820808
NoiseAvg:2.31,Std:0.07,Min:2.1,Max:2.5
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal32.266521
#P14410
Spike_AFFX-r2-Bs-phe_M_signal30.120016
Corner-Avg:6358,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal105.596443
Spike_AFFX-r2-Ec-bioB_3_signal99.897881
Spike_AFFX-r2-Bs-lys_M_signal12.059086
Spike_AFFX-r2-P1-cre_3_signal5380.854492
Spike_AFFX-r2-Bs-lys_3-5-ratio1.482826
Spike_AFFX-r2-Bs-dap_M_signal134.794189
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.219893
Spike_AFFX-r2-Ec-bioB_avg-signal93.691490
Spike_AFFX-r2-Bs-thr_avg-signal67.479256
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal955.356750
Spike_AFFX-r2-Bs-phe_5_signal35.266483
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1410.218872
RawQ2.119932
Spike_AFFX-r2-Bs-lys_5_signal16.474344
Signal(A)5.309330
%A34.888206
Signal(All)152.259735
Corner+Avg:69,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal379.350769
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal34.608128
Spike_AFFX-r2-Ec-bioD_avg-signal1182.787842
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.174046
Spike_AFFX-r2-Bs-lys_avg-signal17.654001
Spike_AFFX-r2-P1-cre_avg-signal4787.113770
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionM
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.937747
Spike_AFFX-r2-Bs-thr_3_signal103.894753
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-signal33.331543
Spike_AFFX-r2-Bs-dap_3_signal172.324509
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.476118
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal309.890289
#M442
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal24.428577
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.224145
Spike_AFFX-r2-Bs-thr_M_signal66.276482
Signal(P)237.554688
Spike_AFFX-r2-Bs-phe_3-5-ratio0.981332
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5186,Count:9
Spike_AFFX-r2-P1-cre_5_signal4193.373047
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7958
Signal(M)17.261948
BackgroundAvg:78.60,Std:0.24,Min:77.6,Max:79.1
Spike_AFFX-r2-Ec-bioC_avg-signal344.620544
Spike_AFFX-r2-Bs-dap_avg-signal132.646500
Spike_AFFX-r2-Bs-dap_3-5-ratio1.897412
Spike_AFFX-r2-Ec-bioB_5_signal75.580147
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.738347
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-8_7-day_pCS19.5_4hr_+CHX -DEX_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 ("Martinez-Garcia_545-8_7-day_pCS19.5_4hr_+CHX -DEX_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: Martinez-Garcia_545-8_7-day_pCS19.5_4hr_+CHX -DEX_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 0uM DEX and 50uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 0uM DEX and 50uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.537013
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-ratio1.012067
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.610781
Spike_AFFX-r2-Bs-dap_5_signal74.256721
NoiseAvg:2.50,Std:0.10,Min:2.2,Max:2.7
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal31.265934
#P15123
Spike_AFFX-r2-Bs-phe_M_signal39.100323
Corner-Avg:13237,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal89.797646
Spike_AFFX-r2-Ec-bioB_3_signal85.574280
Spike_AFFX-r2-Bs-lys_M_signal20.721708
Spike_AFFX-r2-P1-cre_3_signal3120.751465
Spike_AFFX-r2-Bs-lys_3-5-ratio1.412851
Spike_AFFX-r2-Bs-dap_M_signal155.167297
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.142523
Spike_AFFX-r2-Ec-bioB_avg-signal76.165962
Spike_AFFX-r2-Bs-thr_avg-signal63.502518
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal721.102661
Spike_AFFX-r2-Bs-phe_5_signal50.420979
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1175.465088
RawQ2.258438
Spike_AFFX-r2-Bs-lys_5_signal25.837416
Signal(A)4.033598
%A32.091187
Signal(All)145.846191
Corner+Avg:144,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal241.216660
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal46.445068
Spike_AFFX-r2-Ec-bioD_avg-signal948.283875
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P66.299866
Spike_AFFX-r2-Bs-lys_avg-signal27.687851
Spike_AFFX-r2-P1-cre_avg-signal3102.146484
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.608943
Spike_AFFX-r2-Bs-thr_3_signal98.253906
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-signal45.322124
Spike_AFFX-r2-Bs-dap_3_signal181.366714
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.630094
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal208.928513
#M367
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal36.504425
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.154542
Spike_AFFX-r2-Bs-thr_M_signal60.987720
Signal(P)217.680817
Spike_AFFX-r2-Bs-phe_3-5-ratio0.921146
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10970,Count:9
Spike_AFFX-r2-P1-cre_5_signal3083.541260
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7320
Signal(M)14.274573
BackgroundAvg:79.54,Std:0.47,Min:78.8,Max:81.1
Spike_AFFX-r2-Ec-bioC_avg-signal225.072586
Spike_AFFX-r2-Bs-dap_avg-signal136.930237
Spike_AFFX-r2-Bs-dap_3-5-ratio2.442428
Spike_AFFX-r2-Ec-bioB_5_signal53.125957
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.537013
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-9_7-day_pCS19.5_4hr_+CHX -DEX_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 ("Martinez-Garcia_545-9_7-day_pCS19.5_4hr_+CHX -DEX_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: Martinez-Garcia_545-9_7-day_pCS19.5_4hr_+CHX -DEX_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 0uM DEX and 50uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 0uM DEX and 50uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:1.032287
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-ratio1.112409
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.371662
Spike_AFFX-r2-Bs-dap_5_signal70.451088
NoiseAvg:2.22,Std:0.06,Min:2.1,Max:2.5
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal33.897175
#P13922
Spike_AFFX-r2-Bs-phe_M_signal20.753773
Corner-Avg:10819,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal173.090790
Spike_AFFX-r2-Ec-bioB_3_signal127.742607
Spike_AFFX-r2-Bs-lys_M_signal17.849119
Spike_AFFX-r2-P1-cre_3_signal6305.705566
Spike_AFFX-r2-Bs-lys_3-5-ratio2.287747
Spike_AFFX-r2-Bs-dap_M_signal137.970596
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.295873
Spike_AFFX-r2-Ec-bioB_avg-signal131.321060
Spike_AFFX-r2-Bs-thr_avg-signal64.102974
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1418.690796
Spike_AFFX-r2-Bs-phe_5_signal30.040981
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2004.017334
RawQ2.119407
Spike_AFFX-r2-Bs-lys_5_signal17.961859
Signal(A)6.718524
%A36.869793
Signal(All)154.721375
Corner+Avg:118,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal369.099792
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal47.214596
Spike_AFFX-r2-Ec-bioD_avg-signal1711.354004
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.034634
Spike_AFFX-r2-Bs-lys_avg-signal25.634386
Spike_AFFX-r2-P1-cre_avg-signal5987.109375
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_signal77.823624
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-signal32.669785
Spike_AFFX-r2-Bs-dap_3_signal187.698669
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.412582
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal389.008942
#M478
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal41.092182
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.948821
Spike_AFFX-r2-Bs-thr_M_signal80.588112
Signal(P)248.698395
Spike_AFFX-r2-Bs-phe_3-5-ratio1.571673
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:7892,Count:9
Spike_AFFX-r2-P1-cre_5_signal5668.513184
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8410
Signal(M)21.574936
BackgroundAvg:75.45,Std:0.36,Min:74.6,Max:76.8
Spike_AFFX-r2-Ec-bioC_avg-signal379.054382
Spike_AFFX-r2-Bs-dap_avg-signal132.040115
Spike_AFFX-r2-Bs-dap_3-5-ratio2.664241
Spike_AFFX-r2-Ec-bioB_5_signal93.129799
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.032287
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-10_7-day_pCS19.5_4hr_+CHX +DEX_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 ("Martinez-Garcia_545-10_7-day_pCS19.5_4hr_+CHX +DEX_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: Martinez-Garcia_545-10_7-day_pCS19.5_4hr_+CHX +DEX_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 5uM DEX and 50uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 5uM DEX and 50uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.871341
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-ratio1.220538
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.320959
Spike_AFFX-r2-Bs-dap_5_signal102.099899
NoiseAvg:2.42,Std:0.07,Min:2.2,Max:2.7
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal48.694458
#P13911
Spike_AFFX-r2-Bs-phe_M_signal32.440708
Corner-Avg:11621,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal174.068848
Spike_AFFX-r2-Ec-bioB_3_signal111.701324
Spike_AFFX-r2-Bs-lys_M_signal22.085482
Spike_AFFX-r2-P1-cre_3_signal6071.509766
Spike_AFFX-r2-Bs-lys_3-5-ratio1.838017
Spike_AFFX-r2-Bs-dap_M_signal140.989914
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio1.563411
Spike_AFFX-r2-Ec-bioB_avg-signal123.443657
Spike_AFFX-r2-Bs-thr_avg-signal63.570301
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1549.961548
Spike_AFFX-r2-Bs-phe_5_signal31.188753
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1896.700928
RawQ2.193534
Spike_AFFX-r2-Bs-lys_5_signal14.302750
Signal(A)6.151220
%A36.909252
Signal(All)157.156601
Corner+Avg:129,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal398.244385
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal33.383995
Spike_AFFX-r2-Ec-bioD_avg-signal1723.331299
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.986408
Spike_AFFX-r2-Bs-lys_avg-signal20.892311
Spike_AFFX-r2-P1-cre_avg-signal5522.980957
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.104340
Spike_AFFX-r2-Bs-thr_3_signal76.129425
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-signal32.337818
Spike_AFFX-r2-Bs-dap_3_signal167.386917
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.223708
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal365.106018
#M480
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal26.288702
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.090764
Spike_AFFX-r2-Bs-thr_M_signal65.887032
Signal(P)253.309921
Spike_AFFX-r2-Bs-phe_3-5-ratio1.070386
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:8916,Count:9
Spike_AFFX-r2-P1-cre_5_signal4974.452148
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8419
Signal(M)19.084574
BackgroundAvg:79.48,Std:0.27,Min:78.9,Max:80.6
Spike_AFFX-r2-Ec-bioC_avg-signal381.675201
Spike_AFFX-r2-Bs-dap_avg-signal136.825577
Spike_AFFX-r2-Bs-dap_3-5-ratio1.639443
Spike_AFFX-r2-Ec-bioB_5_signal84.560783
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.871341
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-11_7-day_pCS19.5_4hr_+CHX +DEX_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Martinez-Garcia_545-11_7-day_pCS19.5_4hr_+CHX +DEX_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: Martinez-Garcia_545-11_7-day_pCS19.5_4hr_+CHX +DEX_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 5uM DEX and 50uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 5uM DEX and 50uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.752071
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-ratio1.222863
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.411358
Spike_AFFX-r2-Bs-dap_5_signal81.924545
NoiseAvg:2.42,Std:0.06,Min:2.2,Max:2.6
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal30.650543
#P14103
Spike_AFFX-r2-Bs-phe_M_signal29.879099
Corner-Avg:13562,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal163.092194
Spike_AFFX-r2-Ec-bioB_3_signal155.668961
Spike_AFFX-r2-Bs-lys_M_signal16.578737
Spike_AFFX-r2-P1-cre_3_signal6642.205566
Spike_AFFX-r2-Bs-lys_3-5-ratio1.845493
Spike_AFFX-r2-Bs-dap_M_signal117.393219
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.349383
Spike_AFFX-r2-Ec-bioB_avg-signal143.019485
Spike_AFFX-r2-Bs-thr_avg-signal52.302944
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1675.320190
Spike_AFFX-r2-Bs-phe_5_signal34.029678
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2242.058838
RawQ2.182531
Spike_AFFX-r2-Bs-lys_5_signal16.563976
Signal(A)5.645542
%A36.023674
Signal(All)158.964310
Corner+Avg:143,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal463.543365
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal28.121588
Spike_AFFX-r2-Ec-bioD_avg-signal1958.689453
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.828144
Spike_AFFX-r2-Bs-lys_avg-signal21.237137
Spike_AFFX-r2-P1-cre_avg-signal6036.945801
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.148181
Spike_AFFX-r2-Bs-thr_3_signal72.009857
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-signal30.676788
Spike_AFFX-r2-Bs-dap_3_signal165.331818
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.338287
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal388.152313
#M490
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal30.568697
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.194231
Spike_AFFX-r2-Bs-thr_M_signal54.248425
Signal(P)253.163651
Spike_AFFX-r2-Bs-phe_3-5-ratio0.826384
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11401,Count:9
Spike_AFFX-r2-P1-cre_5_signal5431.686035
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8217
Signal(M)18.814955
BackgroundAvg:78.52,Std:0.16,Min:78.3,Max:79.3
Spike_AFFX-r2-Ec-bioC_avg-signal425.847839
Spike_AFFX-r2-Bs-dap_avg-signal121.549866
Spike_AFFX-r2-Bs-dap_3-5-ratio2.018099
Spike_AFFX-r2-Ec-bioB_5_signal110.297318
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.752071
NF1.000000
HZ4
Tau0.015000

Slide: Martinez-Garcia_545-12_7-day_pCS19.5_4hr_+CHX +DEX_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 ("Martinez-Garcia_545-12_7-day_pCS19.5_4hr_+CHX +DEX_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: Martinez-Garcia_545-12_7-day_pCS19.5_4hr_+CHX +DEX_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pCS19
Stock Code:
Genetic Background: Col-0
Age: 7-day old
Growth Conditions:
Stratification3-6 days wrapped in aluminum foil paper at 4ºC
Sterilisation1) Imbibe seeds in 1.5 ml Eppendorf with 1 ml sterile water 0.1% w/v Tween 20 – first vortex a couple of times then 20 minutes on the bench. (2) Remove water, replace with 1 ml 10% w/v sodium hypochlorite 0.1% Tween 20, 10 minutes on the sterile hood (from this point onwards in flow cabinet; do not open tube in lab). (3) Remove hypochlorite solution, wash 5 times with a 1 ml of sterile water. (4) Sow the seeds
ProtocolSeeds were surface sterilized and sown on Petri dishes with GM- (Roig-Villanova et al., 2006). For facilitating harvesting of the seedlings and to avoid shading between them, seeds were sown one by one on filter paper placed on top of GM- medium. After stratification, seeds were germinated and grown in a growth chamber at 22ºC for 7 days under continuous white light (W, 80 umol m-2 s-1 of photosynthetically active radiation; R:FR ratio of 3.2–4.5). On day 7, filter paper was transferred to new plates containing 4 ml of water supplemented or not with 5uM DEX and 50uM CHX. After 4 h seedlings were harvested, frozen in liquid nitrogen and stored at -80ºC for further RNA isolation (Sorin et al., 2009).
ReferenceSorin et al., (2009) Plant J 59, 266-277. Roig-Villanova et al., (2006). Plant Physiol 141, 85-96.
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclave it
Plant Spacing1.64 seedlings per cm2
Temperature22 °C average, 22 °C day, - °C night
Humidity65 % average, 65 % day, - % night
MediumMurashige & Skoog basal salt mixture. Modifications: MES 0.25 g/L pH 5.7-5.8 0.8% bacto-agar
Lighting(Source: Cool white fluorescent manufactured by Phillips. Intensity: 80µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.7
Genetic Variation: T-DNA knock out mutation in At2g44910
Tissue: whole plant
in vivo Treatment: On day 7, seedlings were transferred to new plates containing 4 ml of water supplemented with 5uM DEX and 50uM CHX. After 4 h seedlings were harvested.
Additional Organism Information:
Sample Description7-day-old seedlings
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:0.671982
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-ratio1.217120
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.100941
Spike_AFFX-r2-Bs-dap_5_signal109.850510
NoiseAvg:2.53,Std:0.06,Min:2.3,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal38.466965
#P14451
Spike_AFFX-r2-Bs-phe_M_signal46.622261
Corner-Avg:12998,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal135.488327
Spike_AFFX-r2-Ec-bioB_3_signal100.729530
Spike_AFFX-r2-Bs-lys_M_signal17.675282
Spike_AFFX-r2-P1-cre_3_signal5420.376953
Spike_AFFX-r2-Bs-lys_3-5-ratio1.560164
Spike_AFFX-r2-Bs-dap_M_signal201.397476
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.346435
Spike_AFFX-r2-Ec-bioB_avg-signal109.237282
Spike_AFFX-r2-Bs-thr_avg-signal65.596275
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1189.593628
Spike_AFFX-r2-Bs-phe_5_signal49.424721
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1552.983765
RawQ2.202788
Spike_AFFX-r2-Bs-lys_5_signal24.872248
Signal(A)5.150010
%A34.651470
Signal(All)154.630554
Corner+Avg:150,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal362.330719
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal47.589725
Spike_AFFX-r2-Ec-bioD_avg-signal1371.288696
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.353794
Spike_AFFX-r2-Bs-lys_avg-signal27.117441
Spike_AFFX-r2-P1-cre_avg-signal4936.911133
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.994739
Spike_AFFX-r2-Bs-thr_3_signal90.260246
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-signal47.878906
Spike_AFFX-r2-Bs-dap_3_signal226.025375
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.305474
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal300.267242
#M455
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal38.804798
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.206694
Spike_AFFX-r2-Bs-thr_M_signal68.061607
Signal(P)240.708557
Spike_AFFX-r2-Bs-phe_3-5-ratio0.962873
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10732,Count:9
Spike_AFFX-r2-P1-cre_5_signal4453.445801
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7904
Signal(M)17.445871
BackgroundAvg:82.62,Std:0.26,Min:81.8,Max:83.8
Spike_AFFX-r2-Ec-bioC_avg-signal331.298981
Spike_AFFX-r2-Bs-dap_avg-signal179.091125
Spike_AFFX-r2-Bs-dap_3-5-ratio2.057572
Spike_AFFX-r2-Ec-bioB_5_signal91.494003
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.671982
NF1.000000
HZ4
Tau0.015000


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