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Experiment: Identification of Core Genes Regulating Plant Programmed Cell Death (PCD)
Experiment Description
NASCArrays Experiment Reference Number: NASCARRAYS-77
PCD is a highly organised process that is involved in development and in an organisms response to biotic stresses (toxins and avirulent pathogens) and abiotic stresses (such as temperature, water availability, etc.). It is a genetically regulated form of cellular suicide, however in plants the underlying process is poorly understood. Although PCD may occur in response to different stimuli; we believe once it is triggered, one core mechanism is responsible for the cellular demise. It is our aim to identify the elements of this mechanism. We will do this by expanding on the work of a previous user of GARNet's GeneChip microarray facility, Dr. Jodi Swidzinski. She utilised an Arabidopsis cell suspension system; performing microarray analysis on both senescing, and heat shock induced PCD samples. This resulted in data showing that a large number of genes were upregulated (or downregulated) in response to both treatments. We are working under the premise that some of the genes that were similarly regulated under both treatments must be core PCD genes. However because of the large amount of data generated it is difficult to choose appropriate candidate genes (with any degree of confidence that they are core genes) for further study.
We propose to use a third PCD-inducing treatment, involving a mycotoxin, to generate another population of microarray results. The mycotoxin we intend to use is Fumonisin B1 (FB1). This is an extremely potent compound that induces PCD by disrupting ceramide synthesis. We have found Arabidopsis protoplasts to be much more sensitive than cells to the toxin at low concentrations. Protoplasts are treated with 20mM FB1 and RNA is extracted at time points when 0%, 20% and 40% of protoplasts have died. This RNA is then pooled. RNA from methanol treated protoplasts is used as a control. We intend for these RNA samples to be subjected to GeneChip microarray analysis. This would identify genes differentially regulated due to FB1 treatment, which would be interesting in itself. However, by combining this data with that from previous work by Swidzinski (2002) we will be able to decrease the pool of possible core genes further, and increase the chances of selecting an appropriate candidate gene. This will both improve upon existing work and add value to an existing GARNet data set.
About the ExperimenterName: | Mr. Mark Diamond |
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Head of Lab Name: | Dr. Paul McCabe |
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Lab:
| Botany Department |
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Institute:
| University College Dublin |
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Address: | Botany Department University College Dublin Belfield
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Postcode:
| Dublin 4 |
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Country:
| Ireland |
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| Telephone Number:
| 017162251 |
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Fax Number:
| 017161153 |
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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_treatment_design |
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Number of Slides: | 8 |
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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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no-plants-pooled | 3 cultures |
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biological-replicates | 4 |
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References:
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| Other Information:
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ArrayExpressAccession | E-NASC-22 |
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Slides in this Experiment
Hybridisation Set: Diamond_genome
Slide: Diamond_A-1-Diamo-fum_SLD | | |
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Tissue:
| Cell suspension |
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Diseased:
| Normal |
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in vitro Treatment:
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treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
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Other Information:
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two-or-more-wts | |
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Protocols for BioSource 1 |
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Slide: Diamond_A-2-Diamo-fum_SLD | | |
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Tissue:
| Cell suspension |
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Diseased:
| Normal |
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in vitro Treatment:
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treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
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Other Information:
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two-or-more-wts | |
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Protocols for BioSource 1 |
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Slide: Diamond_A-3-Diamo-fum_SLD | | |
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Tissue:
| Cell suspension |
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Diseased:
| Normal |
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in vitro Treatment:
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treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
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Other Information:
| |
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two-or-more-wts | |
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|
Protocols for BioSource 1 |
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Slide: Diamond_A-4-Diamo-fum_SLD | | |
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Tissue:
| Cell suspension |
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Diseased:
| Normal |
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in vitro Treatment:
| |
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treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
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Other Information:
| |
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two-or-more-wts | |
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|
Protocols for BioSource 1 |
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Slide: Diamond_A-1-Diamo-met_SLD | | |
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Tissue:
| Cell suspension |
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Diseased:
| Normal |
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in vitro Treatment:
| |
---|
treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
---|
Other Information:
| |
---|
two-or-more-wts | |
---|
|
Protocols for BioSource 1 |
|
|
|
Slide: Diamond_A-2-Diamo-met_SLD | | |
|
|
|
Tissue:
| Cell suspension |
---|
Diseased:
| Normal |
---|
in vitro Treatment:
| |
---|
treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
---|
Other Information:
| |
---|
two-or-more-wts | |
---|
|
Protocols for BioSource 1 |
|
|
|
Slide: Diamond_A-3-Diamo-met_SLD | | |
|
|
|
Tissue:
| Cell suspension |
---|
Diseased:
| Normal |
---|
in vitro Treatment:
| |
---|
treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
---|
Other Information:
| |
---|
two-or-more-wts | |
---|
|
Protocols for BioSource 1 |
|
|
|
Slide: Diamond_A-4-Diamo-met_SLD | | |
|
|
|
Tissue:
| Cell suspension |
---|
Diseased:
| Normal |
---|
in vitro Treatment:
| |
---|
treatment | Three, Six day old dark grown cell cultures were pooled and then protoplasted.
Protoplasts were then incubated for 24hrs in a constant temperature room prior
to treatment. Samples were treated with 20uM Fumonisin B1. Samples were frozen
when observed death levels had reached 10%, 20%, and 30%. The RNA was extracted
from these samples. This RNA was then pooled into one sample. |
---|
Other Information:
| |
---|
two-or-more-wts | |
---|
|
Protocols for BioSource 1 |
|
|
|
Problems? Comments? Suggestions? Contact the Affymetrix Team