|
| Status |
Public on Feb 13, 2008 |
| Title |
wat_vs_HrcC_48h_3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NTA0023
|
| Organism |
Nicotiana tabacum |
| Characteristics |
Nicotiana tabacum, Leaf, Biotic stress
|
| Treatment protocol |
Injected with 10^8 CFU/ml Pseudomonas syringae pv. syringae hrcC mutant
|
| Growth protocol |
Grown for 8 weeks in green house (16/8 h light/dark period, 20°C) in soil
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves (SGED_SOP_3.1.1 + SGED_SOP_3.3.1)
|
| Label |
Cy3
|
| Label protocol |
Amino allyl labeling and direct labeling (SGED_SOP_5.1.1 + SGED_SOP_6.1.1)
|
| |
|
| Channel 2 |
| Source name |
NTA0024
|
| Organism |
Nicotiana tabacum |
| Characteristics |
Nicotiana tabacum, Leaf, Biotic stress
|
| Treatment protocol |
Injected with sterile distilled water
|
| Growth protocol |
Grown for 8 weeks in green house (16/8 h light/dark period, 20°C) in soil
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves (SGED_SOP_3.1.1 + SGED_SOP_3.3.1)
|
| Label |
Cy5
|
| Label protocol |
Amino allyl labeling and direct labeling (SGED_SOP_5.1.1 + SGED_SOP_6.1.1)
|
| |
|
| |
| Hybridization protocol |
Hybridization with indirectly labeled mRNA (SGED_SOP_6.1.1, SGED_SOP_6.2.1-4)
|
| Scan protocol |
Axon 4000B scanner. Both the 635nm (red, Cy5) and 532nm (green, Cy3) channels are scanned simultaneously at 100% laser power, the PMT set between 600 and 950. Slides are scanned at a resolution of 10micron
|
| Description |
To study basal resistance we inoculated tobacco plants with Pseudomonas syringae pv. syringae 61 hrcC mutants (gift of A. Collmer). To compare the expression profiles of basal resistance to hypersensitive response, we used P. syringae pv. syringae 61. Gene activation triggered by the live compatible pathogen P. syringae pv. tabaci was compared to kanamycine-inactivated P. tabaci (10 min in 100 ug/ml KM, then washed 2x in H2O). Water-infiltrated control leaves were used. The aim of another set of experiments was to investigate the influence of various signal transduction pathways on basal resistance. Control plants were infiltrated with P. syringae pv syringae hrcC mutant bacteria. These were compared to treatments, in which the bacterial suspensions were supplemented with inhibitors that can block a specific type of signal transduction pathway. The inhibitors represented Ca2+ signaling, lipid derived signals generated by phospholipase C and A, protein phosphorilation (kinases) and protein degradation (degradation of transcription repressors or activators). The used inhibitors were LaCl3, neomycin, aristolochic acid, K252a and MG115 respectively. 2-2.5 months old tobacco plants (Nicotiana tabacum cv. Samsun nn) were grown in a greenhouse in soil with cca. 16/8 hr light/dark period at 20°C, then in a growth chamber 2 days before and during experiments, under identical conditions. A syringe was used for the injection of bacteria or chemical solutions into the tobacco leaves. At 6 or 48 hours post inoculation, leaf samples were taken, and frozen in liquid nitrogen. Every experiment was carried out in 3 independent replicates. Total RNA was extracted using the Quiagen RNeasy Plant Mini kit and the Quiagen Rnase-free Dnase Set, then re-purified and concentrated on Microcon-30 (Millipore) columns. The concentration and quality of isolated RNA was estimated by measuring its absorbency at 260 and 280 nm and running on 1% agarose gel.
|
| Data processing |
The TIFF images were quantified using Genepix 5.0. Local background was subtracted from the signal value and the data was normalized using the quantile method in the limma package of bioconductor.
|
| |
|
| Submission date |
Feb 12, 2008 |
| Last update date |
Feb 12, 2008 |
| Contact name |
Jia Liu |
| E-mail(s) |
[email protected]
|
| URL |
http://www.tigr.org/tdb/potato
|
| Organization name |
Plant Genomics
|
| Street address |
9712 Medical Center Drive
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20850 |
| Country |
USA |
| |
|
| Platform ID |
GPL3838 |
| Series (1) |
| GSE10482 |
Gene expression during innate immunity in tobacco triggered by bacteria. |
|
| Data table header descriptions |
| ID_REF |
Spot identifier for each feature |
| VALUE |
Normalized log2 ratio of normalized intensities defined by CH2/CH1. This value is set to null if it is flagged with "M" or "X" |
| CH1_NORMALIZED |
Normalized background subtracted CH1 intensity (RED channel) |
| CH1_RAW |
Background (CH1_BACKGROUND) subtracted raw intensity (F635 Mean - B635 Media) |
| CH1_BACKGROUND |
CH1 background median intensity (B635 Media) |
| CH2_NORMALIZED |
Normalized background subtracted CH2 intensity (GREEN channel) |
| CH2_RAW |
Background (CH2_BACKGROUND) subtracted raw intensity (F532 Mean - B532 Media) |
| CH2_BACKGROUND |
CH2 background median intensity (B532 Media) |
| FLAG |
B: no flag, good spot; X: undetectable spot; M: flagged for diameter < 70 microns, the percentage of saturated pixels > 30% or not validated PCR product |
