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Experiment: The early post-germinative embryo and endosperm transcriptomes in Arabidopsis

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-386

Arabidopsis seed germination is coordinated with the strong induction of metabolic pathways required for the mobilisation and utilization of seed storage reserves. These are essential to support the seedling before the establishment of photoauxotrophic growth. The activity of genes encoding enzymes required for lipid mobilisation is regulated largely at the level of transcription, but our knowledge of how this regulation occurs is extremely limited. After germination the rate of lipid reserve mobilisation is determined by the carbohydrate status of the seedling and by the osmotic potential of the growth substrate. The plant response to both of these requires the action of the hormone abscisic acid (ABA).

We have shown that this regulation is tissue specific (Penfield et al., 2004 Plant Cell 16, 2705-2718), and that although lipid breakdown in the embryo is inhibited by ABA, lipid breakdown in the endosperm tissues is not. Furthermore, in many species the action of the endosperm is central to the processes controlling seed germination, yet very little is known about gene expression in this tissue, or of the function of the endosperm in mature Arabidopsis seeds. Mature Arabidopsis seeds can be dissected into embryo and endosperm/seed coat fractions, with the latter fraction containing RNA only from the endosperm as the seed coat cells undergo programmed cell death during the latter stages of seed development. In this experiment we divide seeds into embryo and endosperm tissues and transcript profile both shortly after germination, or when germination and lipid reserve mobilisation are inhibited by ABA or by the gibberellin biosynthesis inhibitor paclobutrazol. In this way we can discover the endosperm transcriptome and uncover candidate regulators of lipid mobilisation by searching for genes showing differential expression between embryo and endosperm before and after ABA treatment.

About the Experimenter

Name:Dr Steven Penfield
Head of Lab Name:Prof Ian Graham
Lab: Graham Lab
Institute: Centre for Novel Agricultural Products
Address:Centre for Novel Agricultural Products
Department of Biology
University of York
PO BOX 373
York
Postcode: YO10 5YW
Country: UK
 
Telephone Number: 01904 328759
Fax Number: 01904 328762

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: organism_part_comparison_design
Number of Slides:18
 
Experimental Parameters:
parameterorganism_part
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Penfield: The early post-germinative embryo and endosperm transcriptomes in Arabidopsis_genome

Slide: Penfield_1-1_endosperm-control_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 ("Penfield_1-1_endosperm-control_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: Penfield_1-1_endosperm-control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
LocationGreenhouse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumMurashige and Skoog basal salt mixture
Growth protocolSeeds were sown on MS medium, stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Diseased: Normal
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.895997
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:
RawQ2.43
NoiseAvg:2.84,Stdev:0.06,Max:3.0,Min:2.7
BackgroundAvg:66.16,Stdev:0.34,Max:67.0,Min:65.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.895997
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-2_endosperm-control_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 ("Penfield_1-2_endosperm-control_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: Penfield_1-2_endosperm-control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
LocationGreenhouse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumMurashige and Skoog basal salt mixture
Growth protocolSeeds were sown on MS medium, stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.878637
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:
RawQ2.48
NoiseAvg:2.88,Stdev:0.07,Max:3.2,Min:2.6
BackgroundAvg:68.23,Stdev:1.63,Max:71.4,Min:63.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.878637
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-3_endosperm-control_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 ("Penfield_1-3_endosperm-control_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: Penfield_1-3_endosperm-control_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
LocationGreenhouse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumMurashige and Skoog basal salt mixture
Growth protocolSeeds were sown on MS medium, stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours.
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.595742
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:
RawQ1.87
NoiseAvg:2.13,Stdev:0.08,Max:2.5,Min:2.0
BackgroundAvg:50.87,Stdev:0.49,Max:52.4,Min:49.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.595742
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-4_endosperm-ABA_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 ("Penfield_1-4_endosperm-ABA_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: Penfield_1-4_endosperm-ABA_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumMurashige and Skoog basal salt mixture
Growth protocolSeeds were sown on MS medium supplented with 20uM ABA (mixed iosmers, sigma), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.676544
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:
RawQ2.56
NoiseAvg:3.27,Stdev:0.61,Max:6.9,Min:2.8
BackgroundAvg:69.35,Stdev:0.61,Max:70.0,Min:65.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.676544
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-5_endosperm-ABA_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 ("Penfield_1-5_endosperm-ABA_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: Penfield_1-5_endosperm-ABA_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumMurashige and Skoog basal salt mixture
Growth protocolSeeds were sown on MS medium supplented with 20uM ABA (mixed iosmers, sigma), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.840683
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:
RawQ2.24
NoiseAvg:2.72,Stdev:0.07,Max:2.9,Min:2.5
BackgroundAvg:64.50,Stdev:0.42,Max:66.2,Min:63.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.840683
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-6_endosperm-ABA_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 ("Penfield_1-6_endosperm-ABA_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: Penfield_1-6_endosperm-ABA_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumMurashige and Skoog basal salt mixture
Growth protocolSeeds were sown on MS medium supplented with 20uM ABA (mixed iosmers, sigma), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.567948
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:
RawQ1.97
NoiseAvg:2.22,Stdev:0.09,Max:2.5,Min:2.0
BackgroundAvg:53.13,Stdev:0.44,Max:54.5,Min:51.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.567948
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-7_endosperm-PAC_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 ("Penfield_1-7_endosperm-PAC_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: Penfield_1-7_endosperm-PAC_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplented with 20uM PAC(Greyhound Chromatography, Birkenhead, UK), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.65238
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:
RawQ2.44
NoiseAvg:2.92,Stdev:0.08,Max:3.1,Min:2.7
BackgroundAvg:67.51,Stdev:0.51,Max:68.2,Min:65.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.652380
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-8_endosperm-PAC_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 ("Penfield_1-8_endosperm-PAC_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: Penfield_1-8_endosperm-PAC_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplented with 20uM PAC(Greyhound Chromatography, Birkenhead, UK), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.864509
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:
RawQ2.41
NoiseAvg:2.72,Stdev:0.10,Max:3.0,Min:2.5
BackgroundAvg:63.90,Stdev:0.43,Max:64.9,Min:62.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.864509
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-9_endosperm-PAC_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 ("Penfield_1-9_endosperm-PAC_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: Penfield_1-9_endosperm-PAC_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplented with 20uM PAC(Greyhound Chromatography, Birkenhead, UK), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: endosperm
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.772892
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.10
NoiseAvg:2.31,Stdev:0.06,Max:2.5,Min:2.1
BackgroundAvg:56.40,Stdev:0.56,Max:57.4,Min:54.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.772892
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-10_embryo-control_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 ("Penfield_1-10_embryo-control_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: Penfield_1-10_embryo-control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium, stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.756539
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:
RawQ2.07
NoiseAvg:2.50,Stdev:0.07,Max:2.7,Min:2.2
BackgroundAvg:56.05,Stdev:0.74,Max:57.5,Min:54.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.756539
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-11_embryo-control_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 ("Penfield_1-11_embryo-control_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: Penfield_1-11_embryo-control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium, stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.911314
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:
RawQ2.69
NoiseAvg:3.69,Stdev:1.00,Max:10.2,Min:3.0
BackgroundAvg:71.02,Stdev:0.62,Max:72.2,Min:68.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.911314
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-12_embryo-control_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 ("Penfield_1-12_embryo-control_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: Penfield_1-12_embryo-control_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium, stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.925549
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:
RawQ1.90
NoiseAvg:2.14,Stdev:0.06,Max:2.6,Min:2.0
BackgroundAvg:50.38,Stdev:0.43,Max:51.2,Min:49.0
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.925549
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-13_embryo-ABA_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 ("Penfield_1-13_embryo-ABA_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: Penfield_1-13_embryo-ABA_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplemented with 20uM ABA (mixed isomers, Sigma), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.053806
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:
RawQ2.08
NoiseAvg:2.44,Stdev:0.09,Max:3.0,Min:2.3
BackgroundAvg:56.45,Stdev:0.47,Max:57.5,Min:54.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.053806
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-14_embryo-ABA_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 ("Penfield_1-14_embryo-ABA_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: Penfield_1-14_embryo-ABA_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplemented with 20uM ABA (mixed isomers, Sigma), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.262439
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:
RawQ2.60
NoiseAvg:3.19,Stdev:0.35,Max:5.2,Min:2.8
BackgroundAvg:70.44,Stdev:0.77,Max:71.9,Min:68.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.262439
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-15_embryo-ABA_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 ("Penfield_1-15_embryo-ABA_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: Penfield_1-15_embryo-ABA_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplemented with 20uM ABA (mixed isomers, Sigma), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.848947
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:
RawQ2.15
NoiseAvg:2.38,Stdev:0.16,Max:3.3,Min:2.2
BackgroundAvg:60.29,Stdev:0.36,Max:61.4,Min:59.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.848947
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-16_embryo-PAC_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 ("Penfield_1-16_embryo-PAC_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: Penfield_1-16_embryo-PAC_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplemented with 20uM paclobutrazol (Greyhound chromatography, Birkenhead, UK), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.026304
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:
RawQ2.16
NoiseAvg:2.41,Stdev:0.06,Max:2.6,Min:2.2
BackgroundAvg:57.37,Stdev:0.87,Max:59.4,Min:55.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.026304
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-17_embryo-PAC_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 ("Penfield_1-17_embryo-PAC_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: Penfield_1-17_embryo-PAC_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplemented with 20uM paclobutrazol (Greyhound chromatography, Birkenhead, UK), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:0.614775
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:
RawQ2.71
NoiseAvg:3.25,Stdev:0.09,Max:3.5,Min:3.0
BackgroundAvg:76.20,Stdev:0.55,Max:78.7,Min:75.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.614775
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Penfield_1-18_embryo-PAC_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 ("Penfield_1-18_embryo-PAC_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: Penfield_1-18_embryo-PAC_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
stratification3 days at 4 degrees celsius
sterilisation100% ethanol rinse
Growth substrate type(Source: 0.9% agarose)Agar (100-200 seeds per plate)
Growth mediumSeeds were sown on MS medium supplemented with 20uM paclobutrazol (Greyhound chromatography, Birkenhead, UK), stratified for 3 days at 4 degrees celsius, then tranferred to a growth room at 22 degrees celsius under continuous white light at 80-100 umol m-2 for 24 hours
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)0.50
Tissue: embryo
Additional Organism Information:
Sample descriptionRNA was extracted from manually dissected endosperm samples just after radicle emergence
Separation Technique: manual dissection
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RNA isolation from Arabidopsis seeds
Method:
Required: XT buffer- (for 50mls): 3.82g sodium borate decahydrate (0.2M; MW381.4) 0.57g EGTA (30mM) 0.5g SDS (1%) 0.5g sodium deoxycholate (1%) to pH9.0 with 10M NaOH, DEPC treat and autoclave. If a precipitate forms during storage (after autoclaving) then discard (it will not work if you use it, even if the precipitate is re-dissolved!). Polyvinylpyrrolidone (PVP) Dithiothreitol (DTT) IGEPAL (nonidet P-40, all sigma) 2M Potassium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) 8M Lithium chloride (DEPC treat, or make in sterile falcon tube with DEPC treated water) Proteinase K (PCR grade, Roche) Qiagen Rneasy kit Refridgerated centrifuge, 42oC waterbath, ice bucket
Protocol: 1. To 3.5ml of XT buffer add 0.07g PVP (2% final conc), 5.39g DTT (10mM final conc), IGEPAL to 1% final conc. 2. Grind 130-150 mg seeds (based on dry seed weight) in N2. To frozen pestle and mortar add 500microlitres XT buffer and grind to powder. Decant to 15ml falcon tube (or other). Thaw and at same time add a further 500microlitres XT buffer. [I have also used 70mg seed with 500?l total XT buffer and this too works well, halve all volumes below]. The key to success is step 5. This scales further additions to the volume of the extract rather than the total volume of added buffer, ground material, frozen condensation etc. 3. Add 40microlitres proteinase K, vortex. 4. Incubate tube at 42oC for 90mins. 5. remove 1ml liquid to an eppendorf tube and add 80?l 2M KCl. Mix by inverting. 6. incubate on ice for 60mins. 7. spin at 13000rpm 4oC for 20mins to remove debris. 8. take supernatant to fresh eppendorf, add 360microlitres of 8M LiCl and mix. 9. Precipitate RNA in freezer for at least 2h at -20oC (can be overnight). 10. Collect RNA by spinning at 13000rpm at 4oC for 20mins. 11. Redissolve pellet in 100microlitres Rnase free water. 12. Start RNA cleanup protocol in qiagen handbook: 13. Add beta-mecaptoethanol to RLT buffer (10microlitres per ml). 14. Add 350microlitres RLT buffer to sample. If there is solids in the sample (normal) put through shredder column. 15. Add 250microlitres ethanol, mix by pipetting 16. Apply to Rneasy mini-column (pink) and centrifuge at 13000 for 15secs . 17. Transfer column to new collection tube. Add 500microlitres of RPE buffer, spin for 15secs at 13000rpm. 18. Add a further 500microlitres RPE to column. Spin 1 min at 13000rpm, discard flow-through and spin again for 1 min. 19. Put tube in new collection tube and spin for a further 2mins at 13000rpm. 20. Add 30microlitres Rnase free water to column, leave for 2 mins and elute by spinning for 1min at 13000 rpm. Expected yield from imibibed Arabidopsis seed: 150mg - 30micrograms RNA, 70mg -15micrograms RNA (double is normal for dry seeds).
PublicationSteven Penfield, Eve-Marie Josse, Rubini Kannangara, Alison D. Gilday, Karen J. Halliday and Ian A. Graham, Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA, Current Biology, Volume 15, Issue 22, 22 November 2005, Pages 1998-2006.
Type: total RNA
Other Information:
ProtocolSourceSubmitter

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:1.036041
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:
RawQ1.91
NoiseAvg:2.37,Stdev:0.52,Max:5.8,Min:1.9
BackgroundAvg:50.96,Stdev:0.40,Max:51.7,Min:49.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.036041
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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