|
| Status |
Public on Nov 30, 2012 |
| Title |
Seedlings triple SnRK2 mutant 10 uM ABA 3 hours rep1 vs Seedlings reference Col-0 sample 10 uM ABA 3 hours |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
15 days seedlings Triple SnRK2 mutant rep1 treated with 10 uM ABA during 3 hours
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
material: 15 days seedlings
genotype: snrk2.2/2.3/2.6 triple mutant
treatment: 10 uM ABA during 3 hours
genetic background: Col-0
tissue: seedling
|
| Treatment protocol |
3 hours before harvest samples, all the seedlings were treated with ABA, with final concectration of 10 uM
|
| Growth protocol |
seeds were surface sterilized by treatment with 70% ethanol for 20 min, followed by commercial bleach (2.5 % sodium hypochlorite) containing 0.05 % Triton X-100 for 10 min, and finally, four washes with sterile distilled water. Stratification of the seeds was conducted in the dark at 4ºC for 3 days. Then, seeds were sowed on plates with Murashige-Skoog (MS) solid medium for 4 days. Then they were transfered to sterile flask with MS liquid medium and grown under 100 rpm agitation for 11 days. Plates and flasks were incubated in a controlled environment growth chamber at 22ºC under a 16 h light, 8 h dark photoperiod at 80-100 uE m-2 sec-1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Seedlings were frozen in liquid nitrogen, and grounded in mortars dipped in liquid nitrogen. Total RNA was extracted using the Quiagen Plant RNeasy Mini Kit followong the manufacturer´s recommendations
|
| Label |
Cy5
|
| Label protocol |
0.5 ug of total RNA 0.5 were amplified and labeled with the Agilent Low Input Quick Amp Labeling Kit. Agilent’s Spike-In Kit was used to assess the labeling and hybridization efficiencies
|
| |
|
| Channel 2 |
| Source name |
15 days seedlings Col-0 Reference sample treated with 10 uM ABA during 3 hours
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
material: 15 days seedlings
tissue: seedling
genetic background: Col-0
genotype: wild type Col-0 reference sample
treatment: 10 uM ABA during 3 hours
|
| Treatment protocol |
3 hours before harvest samples, all the seedlings were treated with ABA, with final concectration of 10 uM
|
| Growth protocol |
seeds were surface sterilized by treatment with 70% ethanol for 20 min, followed by commercial bleach (2.5 % sodium hypochlorite) containing 0.05 % Triton X-100 for 10 min, and finally, four washes with sterile distilled water. Stratification of the seeds was conducted in the dark at 4ºC for 3 days. Then, seeds were sowed on plates with Murashige-Skoog (MS) solid medium for 4 days. Then they were transfered to sterile flask with MS liquid medium and grown under 100 rpm agitation for 11 days. Plates and flasks were incubated in a controlled environment growth chamber at 22ºC under a 16 h light, 8 h dark photoperiod at 80-100 uE m-2 sec-1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Seedlings were frozen in liquid nitrogen, and grounded in mortars dipped in liquid nitrogen. Total RNA was extracted using the Quiagen Plant RNeasy Mini Kit followong the manufacturer´s recommendations
|
| Label |
Cy3
|
| Label protocol |
0.5 ug of total RNA 0.5 were amplified and labeled with the Agilent Low Input Quick Amp Labeling Kit. Agilent’s Spike-In Kit was used to assess the labeling and hybridization efficiencies
|
| |
|
| |
| Hybridization protocol |
Hybridization and slide washing were carried out with the Gene Expression Hybridization Kit and Gene Expression Wash Buffers, respectively
|
| Scan protocol |
Scanned on Agilent Technologies Scanner G2505B at a resolution of 5 microns per pixel,using the double scanning as Agilent’s recommended settings. Images were quantified using Agilent Feature Extraction Software (version 9.5.1)
|
| Description |
Triple SnRK2 mutant labelled with Cy5 compared with Col-0 reference sample labelled with Cy3
|
| Data processing |
Inter-array analyses were performed with the GeneSpring 11.5 software. Dye-swap was undo to made all ratio as Col-0 reference data/Mutant data. To ensure high quality dataset, control features were removed, and only features for which the ‘IsWellAboveBG’ parameter was 1 at least in three out of four replicas were selected (31,912 and 31908 features, for pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant and snrk2.2/2.3/2.6 triple mutant analysis, respectively.
|
| |
|
| Submission date |
Mar 21, 2012 |
| Last update date |
Dec 01, 2012 |
| Contact name |
Francisco Vera-Sirera |
| E-mail(s) |
fravesi@ibmcp.upv.es
|
| Organization name |
IBMCP (UPV-CSIC)
|
| Lab |
Lab 2.08
|
| Street address |
Ingeniero Fausto Elio, s/n
|
| City |
Valencia |
| ZIP/Postal code |
46022 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE36692 |
PYR/PYL receptors play a major role for regulation of transcriptional response to abscisic acid |
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