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Experiment: cpl4 microarray analysis

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-658

An Arabidopsis thaliana gene called RNA polymerase II CTD phosphatase-like 4 (CPL4) is orthologous to fungal and metazoan FCP1, and is necessary for normal plant growth and development. RNAi suppression of CPL4 expression results in growth defects that range from transient retardation of seedling development to lethality prior to maturation. The aim of this experiment is to identify genes differentially expressed in CPL4 RNAi plants. For this purpose, seeds were surface sterilized by 70% ethanol and washed with distilled water for several times. Seeds were grown on ¼ strength MS basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. Approximately 200 seeds were sown on each petri dish and stratified for 4 days at 4°C. Then, they were transferred to a growth chamber providing 16h of light and 8h of darkness at 25°C. After 10 days of growth at these conditions, total RNA was extracted from 100mg of seedlings by using TRIzol (Invitrogen, Cat. No. 15596-018) method according to the manufacturer’s procedure. Then, total RNA samples were treated with DNase I (Qiagen, Cat No. 79254) at 37°C for 1 hours and cleaned using Qiagen minicolumns.

About the Experimenter

Name: Hisashi koiwa
Head of Lab Name: Hisashi koiwa
Lab:
Address:Department of Horticultural Sciences, College of Agriculture and Life Science, Texas A
&
M University
202 Horticulture/Forest Science Building, , College Station, Texas, TX 77843-2133, UNITED STATES
Postcode: TX 77843-2133
Country: UNITED STATES
 
Telephone Number: +1 979-845-5282
Fax Number: +1 979-845-0627

All of the data available in this website/database is free, and you are free to do whatever you please with it. If you intend to publish work based on any of this data, please acknowledge us, contact the experimenter above, and either acknowledge them or use them as co-authors in the work.

About this Experiment

Experiment Type: genetic_modification
Number of Slides:6
 
Experimental Parameters:
parametergene_knock_out
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: koiwa: cpl4 microarray analysis

Slide: koiwa_658-1_CPL4-RNAi_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 ("koiwa_658-1_CPL4-RNAi_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: koiwa_658-1_CPL4-RNAi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: cpl4 RNAi line 7-1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
StratificationSeeds were stratified for 4 days at 4°C.
SterilisationSeeds were surface sterilized with 70% ethanol and washed with distilled water for three times.
Other Seed PreparationApproximately 200 seeds were sown on each petri dish
ProtocolSeeds were germinated and grown on 1/4 strength Murashige and Skoog (MS) basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. Plants were grown for 10 days on petri dishes in a growth chamber providing 16h of light and 8h of darkness at 25°C.
Percentage Agrose0.8
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Genetic Variation: RNAi knock out mutation in
Tissue: whole plant
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:0.532768
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-ratio1.271603
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.314537
Spike_AFFX-r2-Bs-dap_5_signal49.567795
NoiseAvg:4.16,Std:0.16,Min:3.7,Max:4.5
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal21.928736
#P14234
Spike_AFFX-r2-Bs-phe_M_signal36.703423
Corner-Avg:12049,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal81.570267
Spike_AFFX-r2-Ec-bioB_3_signal65.601257
Spike_AFFX-r2-Bs-lys_M_signal15.942443
Spike_AFFX-r2-P1-cre_3_signal3983.736084
Spike_AFFX-r2-Bs-lys_3-5-ratio3.319508
Spike_AFFX-r2-Bs-dap_M_signal209.387466
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio4.592397
Spike_AFFX-r2-Ec-bioB_avg-signal65.691994
Spike_AFFX-r2-Bs-thr_avg-signal61.779720
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal674.971252
Spike_AFFX-r2-Bs-phe_5_signal20.292427
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal970.707825
RawQ3.298856
Spike_AFFX-r2-Bs-lys_5_signal17.382967
Signal(A)6.378700
%A35.423061
Signal(All)142.280716
Corner+Avg:497,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal231.666443
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal66.033669
Spike_AFFX-r2-Ec-bioD_avg-signal822.839539
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.402454
Spike_AFFX-r2-Bs-lys_avg-signal30.342768
Spike_AFFX-r2-P1-cre_avg-signal3558.291016
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.174485
Spike_AFFX-r2-Bs-thr_3_signal100.705467
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-signal41.009838
Spike_AFFX-r2-Bs-dap_3_signal385.893738
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.438147
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal218.101913
#M496
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal57.702900
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.062194
Spike_AFFX-r2-Bs-thr_M_signal62.704948
Signal(P)223.599442
Spike_AFFX-r2-Bs-phe_3-5-ratio3.254104
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10852,Count:9
Spike_AFFX-r2-P1-cre_5_signal3132.845947
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8080
Signal(M)22.517841
BackgroundAvg:87.18,Std:1.42,Min:83.6,Max:90.3
Spike_AFFX-r2-Ec-bioC_avg-signal224.884186
Spike_AFFX-r2-Bs-dap_avg-signal214.949661
Spike_AFFX-r2-Bs-dap_3-5-ratio7.785171
Spike_AFFX-r2-Ec-bioB_5_signal49.904472
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.532768
NF1.000000
HZ4
Tau0.015000

Slide: koiwa_658-2_CPL4-RNAi_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 ("koiwa_658-2_CPL4-RNAi_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: koiwa_658-2_CPL4-RNAi_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: cpl4 RNAi line 7-1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
StratificationSeeds were stratified for 4 days at 4°C.
SterilisationSeeds were surface sterilized with 70% ethanol and washed with distilled water for three times.
Other Seed PreparationApproximately 200 seeds were sown on each petri dish
ProtocolSeeds were germinated and grown on 1/4 strength Murashige and Skoog (MS) basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. Plants were grown for 10 days on petri dishes in a growth chamber providing 16h of light and 8h of darkness at 25°C.
Percentage Agrose0.8
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Genetic Variation: RNAi knock out mutation in
Tissue: whole plant
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:0.529702
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-ratio1.277517
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.186975
Spike_AFFX-r2-Bs-dap_5_signal81.797279
NoiseAvg:4.37,Std:0.25,Min:3.7,Max:5.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal33.628448
#P14285
Spike_AFFX-r2-Bs-phe_M_signal42.814247
Corner-Avg:11791,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal69.949730
Spike_AFFX-r2-Ec-bioB_3_signal66.447906
Spike_AFFX-r2-Bs-lys_M_signal27.700121
Spike_AFFX-r2-P1-cre_3_signal3926.166260
Spike_AFFX-r2-Bs-lys_3-5-ratio1.596330
Spike_AFFX-r2-Bs-dap_M_signal264.075775
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio5.773002
Spike_AFFX-r2-Ec-bioB_avg-signal64.126175
Spike_AFFX-r2-Bs-thr_avg-signal106.086029
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal729.618469
Spike_AFFX-r2-Bs-phe_5_signal21.329109
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1077.309570
RawQ3.367401
Spike_AFFX-r2-Bs-lys_5_signal28.658207
Signal(A)6.230256
%A35.366066
Signal(All)141.336197
Corner+Avg:518,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal226.073502
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal55.313370
Spike_AFFX-r2-Ec-bioD_avg-signal903.463989
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.626041
Spike_AFFX-r2-Bs-lys_avg-signal34.035427
Spike_AFFX-r2-P1-cre_avg-signal3499.723145
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.007891
Spike_AFFX-r2-Bs-thr_3_signal194.137085
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal39.818909
Spike_AFFX-r2-Bs-dap_3_signal461.523895
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.476538
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal215.319702
#M458
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal45.747952
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.049943
Spike_AFFX-r2-Bs-thr_M_signal90.492546
Signal(P)221.467529
Spike_AFFX-r2-Bs-phe_3-5-ratio2.593328
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11012,Count:9
Spike_AFFX-r2-P1-cre_5_signal3073.280029
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8067
Signal(M)21.736687
BackgroundAvg:88.49,Std:0.90,Min:86.5,Max:90.8
Spike_AFFX-r2-Ec-bioC_avg-signal220.696594
Spike_AFFX-r2-Bs-dap_avg-signal269.132324
Spike_AFFX-r2-Bs-dap_3-5-ratio5.642289
Spike_AFFX-r2-Ec-bioB_5_signal55.980877
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.529702
NF1.000000
HZ4
Tau0.015000

Slide: koiwa_658-3_CPL4-RNAi_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 ("koiwa_658-3_CPL4-RNAi_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: koiwa_658-3_CPL4-RNAi_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: cpl4 RNAi line 7-1
Stock Code:
Genetic Background: Col-0
Growth Conditions:
StratificationSeeds were stratified for 4 days at 4°C.
SterilisationSeeds were surface sterilized with 70% ethanol and washed with distilled water for three times.
Other Seed PreparationApproximately 200 seeds were sown on each petri dish
ProtocolSeeds were germinated and grown on 1/4 strength Murashige and Skoog (MS) basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. Plants were grown for 10 days on petri dishes in a growth chamber providing 16h of light and 8h of darkness at 25°C.
Percentage Agrose0.8
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Genetic Variation: RNAi knock out mutation in
Tissue: whole plant
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:0.580968
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-ratio1.240618
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.536653
Spike_AFFX-r2-Bs-dap_5_signal103.366333
NoiseAvg:3.82,Std:0.09,Min:3.6,Max:4.2
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal39.801517
#P14436
Spike_AFFX-r2-Bs-phe_M_signal48.239784
Corner-Avg:11151,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal77.550209
Spike_AFFX-r2-Ec-bioB_3_signal67.675713
Spike_AFFX-r2-Bs-lys_M_signal28.712692
Spike_AFFX-r2-P1-cre_3_signal3642.731934
Spike_AFFX-r2-Bs-lys_3-5-ratio1.635576
Spike_AFFX-r2-Bs-dap_M_signal303.079102
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio5.500794
Spike_AFFX-r2-Ec-bioB_avg-signal63.088974
Spike_AFFX-r2-Bs-thr_avg-signal123.910255
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal639.530273
Spike_AFFX-r2-Bs-phe_5_signal21.061504
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal840.551331
RawQ3.167857
Spike_AFFX-r2-Bs-lys_5_signal30.531712
Signal(A)6.142493
%A34.616398
Signal(All)139.882355
Corner+Avg:439,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal228.245926
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal82.232071
Spike_AFFX-r2-Ec-bioD_avg-signal740.040771
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.288033
Spike_AFFX-r2-Bs-lys_avg-signal36.393784
Spike_AFFX-r2-P1-cre_avg-signal3289.477539
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_signal218.939957
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-signal50.511124
Spike_AFFX-r2-Bs-dap_3_signal531.901733
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.314326
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal190.295441
#M478
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal49.936951
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.199429
Spike_AFFX-r2-Bs-thr_M_signal112.989311
Signal(P)216.969894
Spike_AFFX-r2-Bs-phe_3-5-ratio3.904378
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9139,Count:9
Spike_AFFX-r2-P1-cre_5_signal2936.223389
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7896
Signal(M)21.000013
BackgroundAvg:79.90,Std:1.33,Min:77.2,Max:83.7
Spike_AFFX-r2-Ec-bioC_avg-signal209.270691
Spike_AFFX-r2-Bs-dap_avg-signal312.782379
Spike_AFFX-r2-Bs-dap_3-5-ratio5.145793
Spike_AFFX-r2-Ec-bioB_5_signal44.040997
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.580968
NF1.000000
HZ4
Tau0.015000

Slide: koiwa_658-4_gl1-RNAi_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 ("koiwa_658-4_gl1-RNAi_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: koiwa_658-4_gl1-RNAi_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: glabra, virescens, erecta
Stock Code: CS3141
Growth Conditions:
SterilisationSeeds were surface sterilized with 70% ethanol and washed with distilled water for three times.
ProtocolSeeds were grown on 1/4 strength MS basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. After stratification for 4 days at 4°C, plants were grown in a growth chamber providing 16h of light and 8h of darkness at 25°C for 10 days.
Percentage Agrose0.8
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Tissue: whole plant
Additional Organism Information:
Sample Description10 day old whole seedlings were used for RNA extraction
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:0.75377
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-ratio1.245313
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.239122
Spike_AFFX-r2-Bs-dap_5_signal69.526283
NoiseAvg:3.44,Std:0.11,Min:3.3,Max:4.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal32.050873
#P14114
Spike_AFFX-r2-Bs-phe_M_signal40.154057
Corner-Avg:9969,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal102.416740
Spike_AFFX-r2-Ec-bioB_3_signal74.052231
Spike_AFFX-r2-Bs-lys_M_signal16.545607
Spike_AFFX-r2-P1-cre_3_signal4838.564453
Spike_AFFX-r2-Bs-lys_3-5-ratio1.799068
Spike_AFFX-r2-Bs-dap_M_signal226.058914
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio4.312994
Spike_AFFX-r2-Ec-bioB_avg-signal78.743614
Spike_AFFX-r2-Bs-thr_avg-signal82.442017
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal891.302551
Spike_AFFX-r2-Bs-phe_5_signal22.549335
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1277.789429
RawQ3.018081
Spike_AFFX-r2-Bs-lys_5_signal26.256950
Signal(A)7.105925
%A35.975449
Signal(All)146.019104
Corner+Avg:413,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal304.942200
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal58.861862
Spike_AFFX-r2-Ec-bioD_avg-signal1084.546021
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.876369
Spike_AFFX-r2-Bs-lys_avg-signal30.013535
Spike_AFFX-r2-P1-cre_avg-signal4361.992188
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_signal138.235245
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-signal40.521748
Spike_AFFX-r2-Bs-dap_3_signal403.328491
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.433620
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal283.789795
#M490
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal47.238049
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.074535
Spike_AFFX-r2-Bs-thr_M_signal77.039932
Signal(P)231.019272
Spike_AFFX-r2-Bs-phe_3-5-ratio2.610359
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9423,Count:9
Spike_AFFX-r2-P1-cre_5_signal3885.420410
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8206
Signal(M)24.038021
BackgroundAvg:74.74,Std:0.55,Min:73.7,Max:76.7
Spike_AFFX-r2-Ec-bioC_avg-signal294.365997
Spike_AFFX-r2-Bs-dap_avg-signal232.971237
Spike_AFFX-r2-Bs-dap_3-5-ratio5.801094
Spike_AFFX-r2-Ec-bioB_5_signal59.761868
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.753770
NF1.000000
HZ4
Tau0.015000

Slide: koiwa_658-5_gl1-RNAi_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 ("koiwa_658-5_gl1-RNAi_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: koiwa_658-5_gl1-RNAi_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: glabra, virescens, erecta
Stock Code: CS3141
Growth Conditions:
SterilisationSeeds were surface sterilized with 70% ethanol and washed with distilled water for three times.
ProtocolSeeds were grown on 1/4 strength MS basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. After stratification for 4 days at 4°C, plants were grown in a growth chamber providing 16h of light and 8h of darkness at 25°C for 10 days.
Percentage Agrose0.8
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Tissue: whole plant
Additional Organism Information:
Sample Description10 day old whole seedlings were used for RNA extraction
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:0.612616
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-ratio1.235520
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.267231
Spike_AFFX-r2-Bs-dap_5_signal43.465176
NoiseAvg:3.71,Std:0.16,Min:3.3,Max:4.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal15.274648
#P14486
Spike_AFFX-r2-Bs-phe_M_signal33.800144
Corner-Avg:10404,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal88.826309
Spike_AFFX-r2-Ec-bioB_3_signal73.679047
Spike_AFFX-r2-Bs-lys_M_signal14.297994
Spike_AFFX-r2-P1-cre_3_signal4125.222168
Spike_AFFX-r2-Bs-lys_3-5-ratio2.626556
Spike_AFFX-r2-Bs-dap_M_signal172.332932
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio7.696370
Spike_AFFX-r2-Ec-bioB_avg-signal73.549034
Spike_AFFX-r2-Bs-thr_avg-signal62.293499
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal761.850769
Spike_AFFX-r2-Bs-phe_5_signal18.254414
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1187.555176
RawQ3.041312
Spike_AFFX-r2-Bs-lys_5_signal20.101898
Signal(A)6.362789
%A34.462955
Signal(All)143.703308
Corner+Avg:397,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal255.662354
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal52.010418
Spike_AFFX-r2-Ec-bioD_avg-signal974.703003
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.507233
Spike_AFFX-r2-Bs-lys_avg-signal29.066216
Spike_AFFX-r2-P1-cre_avg-signal3732.038574
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.029811
Spike_AFFX-r2-Bs-thr_3_signal117.559341
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-signal34.688324
Spike_AFFX-r2-Bs-dap_3_signal297.995575
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.558777
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal233.073547
#M463
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal52.798756
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.096917
Spike_AFFX-r2-Bs-thr_M_signal54.046513
Signal(P)222.113861
Spike_AFFX-r2-Bs-phe_3-5-ratio2.849197
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9731,Count:9
Spike_AFFX-r2-P1-cre_5_signal3338.854736
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7861
Signal(M)22.274517
BackgroundAvg:76.96,Std:0.61,Min:75.7,Max:78.9
Spike_AFFX-r2-Ec-bioC_avg-signal244.367950
Spike_AFFX-r2-Bs-dap_avg-signal171.264542
Spike_AFFX-r2-Bs-dap_3-5-ratio6.855962
Spike_AFFX-r2-Ec-bioB_5_signal58.141750
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.612616
NF1.000000
HZ4
Tau0.015000

Slide: koiwa_658-6_gl1-RNAi_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 ("koiwa_658-6_gl1-RNAi_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: koiwa_658-6_gl1-RNAi_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: glabra, virescens, erecta
Stock Code: CS3141
Growth Conditions:
SterilisationSeeds were surface sterilized with 70% ethanol and washed with distilled water for three times.
ProtocolSeeds were grown on 1/4 strength MS basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. After stratification for 4 days at 4°C, plants were grown in a growth chamber providing 16h of light and 8h of darkness at 25°C for 10 days.
Percentage Agrose0.8
Temperature25 °C average, 25 °C day, 25 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture
Lighting
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Tissue: whole plant
Additional Organism Information:
Sample Description10 day old whole seedlings were used for RNA extraction
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:0.667469
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-ratio1.253323
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.447923
Spike_AFFX-r2-Bs-dap_5_signal39.109528
NoiseAvg:3.84,Std:0.07,Min:3.6,Max:4.0
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal25.291792
#P14244
Spike_AFFX-r2-Bs-phe_M_signal24.004877
Corner-Avg:10688,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal96.061378
Spike_AFFX-r2-Ec-bioB_3_signal76.616486
Spike_AFFX-r2-Bs-lys_M_signal23.466871
Spike_AFFX-r2-P1-cre_3_signal4547.160156
Spike_AFFX-r2-Bs-lys_3-5-ratio1.258035
Spike_AFFX-r2-Bs-dap_M_signal138.111404
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.389589
Spike_AFFX-r2-Ec-bioB_avg-signal75.197540
Spike_AFFX-r2-Bs-thr_avg-signal59.354477
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal912.719788
Spike_AFFX-r2-Bs-phe_5_signal20.979254
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1381.500244
RawQ3.196860
Spike_AFFX-r2-Bs-lys_5_signal18.965870
Signal(A)6.735703
%A35.453747
Signal(All)145.568558
Corner+Avg:449,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal273.969727
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal39.080055
Spike_AFFX-r2-Ec-bioD_avg-signal1147.109985
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.446297
Spike_AFFX-r2-Bs-lys_avg-signal22.097494
Spike_AFFX-r2-P1-cre_avg-signal4087.622070
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.099956
Spike_AFFX-r2-Bs-thr_3_signal85.728775
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-signal28.021395
Spike_AFFX-r2-Bs-dap_3_signal265.792786
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.513608
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal264.964081
#M479
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal23.859734
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.033988
Spike_AFFX-r2-Bs-thr_M_signal67.042862
Signal(P)228.501511
Spike_AFFX-r2-Bs-phe_3-5-ratio1.862795
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9873,Count:9
Spike_AFFX-r2-P1-cre_5_signal3628.083984
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8087
Signal(M)23.323069
BackgroundAvg:81.76,Std:0.79,Min:80.0,Max:83.7
Spike_AFFX-r2-Ec-bioC_avg-signal269.466919
Spike_AFFX-r2-Bs-dap_avg-signal147.671234
Spike_AFFX-r2-Bs-dap_3-5-ratio6.796113
Spike_AFFX-r2-Ec-bioB_5_signal52.914761
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.667469
NF1.000000
HZ4
Tau0.015000


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