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Experiment: Analysis of RLI on RNA stability in A. thaliana

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-460

A number of RNA encoding different functions have been detected in the plant vasculature. An RNase L inhibitor, similar to that present in humans have been identified in the phloem sap of cucurbits. We are interested in analyze the role of this putative inhibitor on the transcript levels that could be present in the vasculature and mainly in the phloem stream. For this purpose, we obtained an RLI T-DNA insertion mutant and generated A. thaliana overexpressing lines using the ORF of the two alleles found in the database.

About the Experimenter

Name:Dr Beatriz Xoconostle
Head of Lab Name:Dr Beatriz Xoconostle
Lab:
Address:Biotecnologia Cinvestav
Ave. IPN 2508
San Pedro Zacatenco
Mexico City D.F.
Postcode: 07360
Country: Mexico
 
Telephone Number: 525557473800
Fax Number: 5255557473313

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_design
Number of Slides:6
 
Experimental Parameters:
parametergene_knock_out
Quality Control Measures Taken:
no-plants-pooled
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Xoconostle: Analysis of RLI on RNA stability in A. thaliana_genome

Slide: Xoconostle_2-1_WT1_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 ("Xoconostle_2-1_WT1_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: Xoconostle_2-1_WT1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Co-1
Stock Code: N1084
Age: 26 days
Growth Conditions:
sterilisationWe sterilized the seeds using ethanol 50% for 10 minutes.
Growth protocolSeeds were sterilized, mixed with agarose 0.01% and dropped onto prewet sunshine mix soil. Trays were covered until plantelets appeared.
LocationGrowth chamber
Growth substrate typeCommercial soil
Spacing4 plants/pot 4in2
Average humidity70%
Average temperature26°C
growthmediumHoagland's No.2 basal salt mixture
Sample descriptionWe used the whole plant. Once extracted carefully from the soil, the root was rinsed with distilled water in order to remove the soil.
Developmental Stage: 3.20
Tissue: whole plant
Other Information:
RNAQCNo
RNAReceivedNo

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:1.275817632675
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.32
NoiseAvg:2.27,Stdev:0.06,Max:2.5,Min:2.1
Central-Avg:12029,Count:9
Corner+Avg:97,Count:32
Corner-Avg:12844,Count:32
BackgroundAvg:54.71,Stdev:0.33,Max:55.3,Min:53.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.275817632675
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Xoconostle_2-2_WT2_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 ("Xoconostle_2-2_WT2_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: Xoconostle_2-2_WT2_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Co-1
Stock Code: N1084
Age: 26 days
Growth Conditions:
sterilisationWe sterilized the seeds using ethanol 50% for 10 minutes.
Growth protocolSeeds were sterilized, mixed with agarose 0.01% and dropped onto prewet sunshine mix soil. Trays were covered until plantelets appeared.
LocationGrowth chamber
Growth substrate typeCommercial soil
Spacing4 plants/pot 4in2
Average humidity70%
Average temperature26°C
growthmediumHoagland's No.2 basal salt mixture
Sample descriptionWe used the whole plant. Once extracted carefully from the soil, the root was rinsed with distilled water in order to remove the soil.
Developmental Stage: 3.20
Tissue: whole plant
Other Information:
RNAQCNo
RNAReceivedNo

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:1.320522546768
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.29
NoiseAvg:2.25,Stdev:0.05,Max:2.4,Min:2.1
Central-Avg:13392,Count:9
Corner+Avg:106,Count:32
Corner-Avg:14248,Count:32
BackgroundAvg:53.59,Stdev:0.52,Max:54.6,Min:51.9
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.320522546768
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Xoconostle_2-3_WT3_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 ("Xoconostle_2-3_WT3_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: Xoconostle_2-3_WT3_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Co-1
Stock Code: N1084
Age: 26 days
Growth Conditions:
sterilisationWe sterilized the seeds using ethanol 50% for 10 minutes.
Growth protocolSeeds were sterilized, mixed with agarose 0.01% and dropped onto prewet sunshine mix soil. Trays were covered until plantelets appeared.
LocationGrowth chamber
Growth substrate typeCommercial soil
Spacing4 plants/pot 4in2
Average humidity70%
Average temperature26°C
growthmediumHoagland's No.2 basal salt mixture
Sample descriptionWe used the whole plant. Once extracted carefully from the soil, the root was rinsed with distilled water in order to remove the soil.
Developmental Stage: 3.20
Tissue: whole plant
Other Information:
RNAQCNo
RNAReceivedNo

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:3.162996292114
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.85
NoiseAvg:2.88,Stdev:0.16,Max:3.8,Min:2.7
Central-Avg:11163,Count:9
Corner+Avg:84,Count:32
Corner-Avg:10916,Count:32
BackgroundAvg:67.86,Stdev:0.72,Max:69.6,Min:65.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF3.162996292114
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Xoconostle_2-4_rli-1_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 ("Xoconostle_2-4_rli-1_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: Xoconostle_2-4_rli-1_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Co-1
Stock Code: N1084
Age: 26 days
Growth Conditions:
sterilisationWe sterilized the seeds using ethanol 50% for 10 minutes.
Growth protocolSeeds were sterilized, mixed with agarose 0.01% and dropped onto prewet sunshine mix soil. Trays were covered until plantelets appeared.
LocationGrowth chamber
Growth substrate typeCommercial soil
Spacing4 plants/pot 4in2
Average humidity70%
Average temperature26°C
growthmediumHoagland's No.2 basal salt mixture
Sample descriptionWe used the whole plant. Once extracted carefully from the soil, the root was rinsed with distilled water in order to remove the soil.
Developmental Stage: 1.14
Tissue: whole plant
Other Information:
RNAQCNo
RNAReceivedNo

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:2.576572656631
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.42
NoiseAvg:2.28,Stdev:0.15,Max:2.9,Min:1.9
Central-Avg:10083,Count:9
Corner+Avg:82,Count:32
Corner-Avg:11194,Count:32
BackgroundAvg:55.36,Stdev:2.05,Max:60.4,Min:51.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.576572656631
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Xoconostle_2-5_rli-2_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 ("Xoconostle_2-5_rli-2_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: Xoconostle_2-5_rli-2_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Co-1
Stock Code: N1084
Age: 26 days
Growth Conditions:
sterilisationWe sterilized the seeds using ethanol 50% for 10 minutes.
Growth protocolSeeds were sterilized, mixed with agarose 0.01% and dropped onto prewet sunshine mix soil. Trays were covered until plantelets appeared.
LocationGrowth chamber
Growth substrate typeCommercial soil
Spacing4 plants/pot 4in2
Average humidity70%
Average temperature26°C
growthmediumHoagland's No.2 basal salt mixture
Sample descriptionWe used the whole plant. Once extracted carefully from the soil, the root was rinsed with distilled water in order to remove the soil.
Developmental Stage: 1.14
Tissue: whole plant
Other Information:
RNAQCNo
RNAReceivedNo

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:1.697477936745
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.22
NoiseAvg:2.16,Stdev:0.08,Max:2.4,Min:2.0
Central-Avg:10945,Count:9
Corner+Avg:80,Count:32
Corner-Avg:11266,Count:32
BackgroundAvg:54.32,Stdev:0.65,Max:55.6,Min:53.2
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.697477936745
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Xoconostle_2-6_rli-3_Rep3_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 ("Xoconostle_2-6_rli-3_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: Xoconostle_2-6_rli-3_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Co-1
Stock Code: N1084
Age: 26 days
Growth Conditions:
sterilisationWe sterilized the seeds using ethanol 50% for 10 minutes.
Growth protocolSeeds were sterilized, mixed with agarose 0.01% and dropped onto prewet sunshine mix soil. Trays were covered until plantelets appeared.
LocationGrowth chamber
Growth substrate typeCommercial soil
Spacing4 plants/pot 4in2
Average humidity70%
Average temperature26°C
growthmediumHoagland's No.2 basal salt mixture
Sample descriptionWe used the whole plant. Once extracted carefully from the soil, the root was rinsed with distilled water in order to remove the soil.
Developmental Stage: 1.14
Tissue: whole plant
Other Information:
RNAQCNo
RNAReceivedNo

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:2.133706331253
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.40
NoiseAvg:2.36,Stdev:0.07,Max:2.6,Min:2.2
Central-Avg:11080,Count:9
Corner+Avg:85,Count:32
Corner-Avg:11342,Count:32
BackgroundAvg:58.27,Stdev:1.22,Max:60.4,Min:54.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF2.133706331253
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


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