|
| Status |
Public on Apr 02, 2010 |
| Title |
AP1-GRap1cal_2hr_Dex vs. Mock_set3_4000B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
AP1-GRap1cal_2hr Dex_set3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genome/variation: AP1-GR ap1 cal inflorescences
treatment group: Dex
treatment time: 2hr
|
| Treatment protocol |
For all experiments, we used approximately 4 week-old 35S:AP1-GR ap1-1 cal-1 plants. For each sample, inflorescence tissue from ~25 plants was collected using jeweler’s forceps. Four biologically independent sets of samples were generated. Inflorescences were treated with a solution containing 10 μM dexamethasone (Sigma-Aldrich), 0.01% (v/v) ethanol, and 0.015% (v/v) Silwett L-77 (De Sangosse), or with an identical mock solution that lacked dexamethasone. We then collected inflorescence tissue after 2, 4, 8, and 12 hours, as well as from untreated plants (0 hour time point).
|
| Growth protocol |
Plants were grown on a soil:vermiculite:perlite (2:1:1) mixture at 20ºC under constant illumination with cool white fluorescent light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from tissue samples using the Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer’s instructions. Quality of RNA samples was evaluated using a Bioanalyzer and a RNA Nano 6000 kit (Agilent). RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| Label |
Cy3
|
| Label protocol |
Dye-labeled antisense RNA was generated from the total RNA preparation. The Amino Allyl MessageAmpII aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX) was used for labeling of RNA samples with Cy3 and Cy5 fluorescent dyes, following manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
AP1-GRap1cal_2hr Mock_set3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genome/variation: AP1-GR ap1 cal inflorescences
treatment group: Mock
treatment time: 2hr
|
| Treatment protocol |
For all experiments, we used approximately 4 week-old 35S:AP1-GR ap1-1 cal-1 plants. For each sample, inflorescence tissue from ~25 plants was collected using jeweler’s forceps. Four biologically independent sets of samples were generated. Inflorescences were treated with a solution containing 10 μM dexamethasone (Sigma-Aldrich), 0.01% (v/v) ethanol, and 0.015% (v/v) Silwett L-77 (De Sangosse), or with an identical mock solution that lacked dexamethasone. We then collected inflorescence tissue after 2, 4, 8, and 12 hours, as well as from untreated plants (0 hour time point).
|
| Growth protocol |
Plants were grown on a soil:vermiculite:perlite (2:1:1) mixture at 20ºC under constant illumination with cool white fluorescent light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from tissue samples using the Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer’s instructions. Quality of RNA samples was evaluated using a Bioanalyzer and a RNA Nano 6000 kit (Agilent). RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| Label |
Cy5
|
| Label protocol |
Dye-labeled antisense RNA was generated from the total RNA preparation. The Amino Allyl MessageAmpII aRNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX) was used for labeling of RNA samples with Cy3 and Cy5 fluorescent dyes, following manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
Dye-labeled antisense RNA was hybridized to microarrays using a MAUI hybridization system (BioMicro Systems, Salt Lake City, UT, USA) as previously described (Wellmer et al. (2006) PLoS Genetics 2, e117). Specifically, hybridizations were done as follows: dye-labeled antisense RNA preparations were dried down and the resulting pellets were re-suspended in 5 ul 10 mM EDTA and 45 ul SlideHyb Buffer #1 (Ambion, Austin, Texas, United States) and hybridized for 14 h to microarrays at 48C using a MAUI hybridization system (BioMicro Systems, Salt Lake City, Utah, United States) according to the manufacturer's instructions.
|
| Scan protocol |
Microarrays were scanned and the data acquired with an Axon GenePix 4000B scanner using GenePix Pro 5.1.0.19 analysis software.
|
| Description |
s42_AP1GRap1cal_2hDexMock_G400R600_set3_JLR4000B.gpr
|
| Data processing |
Data were processed with the Resolver version 7.1 data analysis system (Rosetta Biosoftware, Seattle, WA), using the Axon/GenePix Ratio data processing pipeline with default settings. Resolver uses an error model-based approach to stabilize the variance estimation to improve the specificity and sensitivity in differential gene expression detection (Weng, L., Dai, H., Zhan, Y., He, Y., Stepaniants, S.B., and Bassett, D.E. (2006). Rosetta error model for gene expression analysis. Bioinformatics 22, 1111-1121).
|
| |
|
| Submission date |
Feb 03, 2010 |
| Last update date |
Jun 14, 2013 |
| Contact name |
Jose Luis Riechmann |
| E-mail(s) |
joseluis.riechmann@cragenomica.es
|
| Organization name |
Center for Research in Agricultural Genomics
|
| Street address |
Campus UAB - Edifici CRAG
|
| City |
Bellaterra |
| State/province |
Barcelona |
| ZIP/Postal code |
08193 |
| Country |
Spain |
| |
|
| Platform ID |
GPL9989 |
| Series (2) |
| GSE20183 |
Meyerowitz Lab Arabidopsis Operon Array v4 - 4000B scanner - AP1-GR ap1cal inflorescences - time series (hours) |
| GSE20184 |
Orchestration of floral initiation by APETALA1 |
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