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Experiment: Identification of genes that are directly regulated by NMD in Arabidopsis
Experiment Description
NASCArrays Experiment Reference Number: NASCARRAYS-647
To understand the way NMD mediates responses it is important to distinguish between direct and indirect targets. We will accomplish this using a global analysis of mRNA stability in wild type and NMD mutant backgrounds. Direct NMD targets show enhanced stability exclusively in NMD mutants. We will use cordycepin to interrupt transcription in wild type and NMD mutant three week old seedlings and analyse samples at time intervals following treatment. We will take timepoints at 0hrs, 1hr, 3hrs and 6hrs. We will analyse global mRNA stability in seedlings of four of our NMD mutant lines (upf1-5, UPF2 (6-11E5), upf3-1 and smg7-1) and wild type. This experiment will generate a list of mRNAs that are directly destabilised by NMD, which can be compared and analysed for features that make them targets of NMD. Three crucial questions will be addressed by this experiment. Firstly, the mRNA decay data will allow us to identify the genes that are directly targeted by each subset of the NMD pathway. For example, targets that are independent of UPF3 will appear to have altered stability only in the upf1 and smg7 mutant backgrounds. Secondly, it will allow us to test the association of specific NMD trigger signals with subsets of NMD. Finally, the list of direct targets will facilitate identification and dissection of the downstream regulated processes.
About the ExperimenterName: | Dr Samantha Rayson |
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Head of Lab Name: | Dr Brendan Davies |
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Lab:
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Address: | Centre for Plant Sciences, Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds Leeds, West Yorkshire, LS2 9JT, UNITED KINGDOM
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Postcode:
| LS2 9JT |
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Country:
| UNITED KINGDOM |
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| Telephone Number:
| 011334332838 |
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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.
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| About this ExperimentExperiment Type:
| compound_based_treatment |
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Number of Slides: | 20 |
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| Experimental Parameters:
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parameter | compound_based_treatment |
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Quality Control Measures Taken:
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References:
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| Other Information:
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Slides in this Experiment
Hybridisation Set: Rayson: Identification of genes that are directly regulated by NMD in Arabidopsis
Slide: Rayson_647-9_upf1-5_NMD_0hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 0
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-8_smg7-1_NMD_6hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 6 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0.
One leaf was taken for genotyping 1 week prior to treatment. Only confirmed homozygotes were used. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-7_smg7-1_NMD_3hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 3 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0.
One leaf was taken for genotyping 1 week prior to treatment. Only confirmed homozygotes were used. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-6_smg7-1_NMD_1hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 1hour
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0.
One leaf was taken for genotyping 1 week prior to treatment. Only confirmed homozygotes were used. |
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Additional Organism Information:
| |
---|
Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-5_smg7-1_NMD_0hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 0
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0.
One leaf was taken for genotyping 1 week prior to treatment. Only confirmed homozygotes were used. |
---|
Additional Organism Information:
| |
---|
Sample Description | Soil-grown plant grown under short-day conditions. |
---|
Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-4_WT_6hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 6 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-3_WT_3hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 3 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-2_WT_1hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 1 hour
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-1_WT_0hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 0 (no cordycepin)
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-20_upf3-1_NMD_6hr _cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 6 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Slide: Rayson_647-19_upf3-1_NMD_3hr _cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 3 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Slide: Rayson_647-18_upf3-1_NMD_1hr _cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 1 hour
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Slide: Rayson_647-17_upf3-1_NMD_0hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 0
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-16_UPF2(6-11E5)_NMD_6hr _cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 6 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Slide: Rayson_647-12_upf1-5_NMD_6hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 6 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Slide: Rayson_647-11_upf1-5_NMD_3hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 3 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-10_upf1-5_NMD_1hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 1 hour
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-15_UPF2(6-11E5)_NMD_3hr _cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 3 hours
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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Protocols for BioSource 1 |
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Slide: Rayson_647-14_UPF2(6-11E5)_NMD_1hr _cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 1 hour
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
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Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Slide: Rayson_647-13_UPF2(6-11E5)_NMD_0hr_cordycepin_Rep1_ATH1 | | |
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Tissue:
| Aerial tissues |
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in vivo Treatment:
| Time = 0
Aerial tissues were harvested into incubation buffer (IB: 1 mM Pipes, 1 mM Sodium citrate, 1 mM Potassium chloride, 15 mM Sucrose). After 30 minutes in IB, all except the time (T) = 0 samples were transferred to IB with cordycepin (150 µg ml-1). The T = 0 samples were transferred to fresh IB without cordycepin. All flasks (including the T = 0) were transferred to a bell jar and vacuum infiltrated. Immediately post-infiltration was classed as T =0. |
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Additional Organism Information:
| |
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Sample Description | Soil-grown plant grown under short-day conditions. |
---|
Other Information:
| |
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Timecourse start procedure | The time-course started immediately after the vacuum infiltration. |
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|
Protocols for BioSource 1 |
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Problems? Comments? Suggestions? Contact the Affymetrix Team