|
| Status |
Public on Apr 25, 2012 |
| Title |
PXVE:NF-YA2SRDX 24h estradiol rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
complete 8-day-old seedlings estradiol-exposed for 24 h
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genetic background: Columbia 0
genotype: PXVE:NF-YA2SRDX
age: 8 days
tissue: whole seedling
treatment: 7 uM estradiol
|
| Treatment protocol |
Eight-day-old seedlings grown on liquid medium were exposed for 24 h to 7 mM b-estradiol or DMSO (mock treatment) prior to RNA extraction
|
| Growth protocol |
Arabidopsis thaliana ecotype Col-0 seeds were surface sterilized with 70% (v/v) ethanol for 7 min and 20% (v/v) bleach for 7 min. After three washes in distilled water, seeds were germinated and grown on agar plates containing 0.1X Murashige and Skook medium. Plates were placed vertically at an angle of 65˚ to allow root growth along the agar surface and to allow unimpeded growth of the hypocotyl into the air. For plant growth, we used a plant growth cabinet (Percival Scientific AR95L, Perry, IA), with a photoperiod of 16 h of light, 8 h of darkness, and temperature of 22 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from whole seedlings using the RNeasy kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
For each transgenic line, three biological replicates were used for probe synthesis. For each sample, 1.5 μg total RNA were amplified in the presence of aminoallyl-dUTP (Ambion) using the Aminoallyl Message Amp II kit (Ambion). Resulting amplified RNA probes were further labeled with fluorescent Cy3 and Cy5 dyes (Amersham). The fluorescent dye-labeled probes were then purified using RNeasy columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Total RNA from pooled PXVE transgenic lines DMSO-exposed for 24h
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genetic background: Columbia 0
genotype: Pooled PXVE transgenic lines
age: 8 days
tissue: whole seedling
treatment: DMSO
|
| Treatment protocol |
Eight-day-old seedlings grown on liquid medium were exposed for 24 h to 7 mM b-estradiol or DMSO (mock treatment) prior to RNA extraction
|
| Growth protocol |
Arabidopsis thaliana ecotype Col-0 seeds were surface sterilized with 70% (v/v) ethanol for 7 min and 20% (v/v) bleach for 7 min. After three washes in distilled water, seeds were germinated and grown on agar plates containing 0.1X Murashige and Skook medium. Plates were placed vertically at an angle of 65˚ to allow root growth along the agar surface and to allow unimpeded growth of the hypocotyl into the air. For plant growth, we used a plant growth cabinet (Percival Scientific AR95L, Perry, IA), with a photoperiod of 16 h of light, 8 h of darkness, and temperature of 22 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from whole seedlings using the RNeasy kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
For each transgenic line, three biological replicates were used for probe synthesis. For each sample, 1.5 μg total RNA were amplified in the presence of aminoallyl-dUTP (Ambion) using the Aminoallyl Message Amp II kit (Ambion). Resulting amplified RNA probes were further labeled with fluorescent Cy3 and Cy5 dyes (Amersham). The fluorescent dye-labeled probes were then purified using RNeasy columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Probes were mixed, concentrated by precipitation and resuspended in the hybridization solution (50% formamide, 5X SSC, 0.1% SDS, 0.4 μg/ l tRNA and 0.2 μg/ l Salmon Sperm DNA) for 14h. Slides were washed 5 min in each of the following solutions: a) 2x SSC, 0.1% SDS/42°C, b) 0.1x SSC/RT and two final washes with 0.05x SSC/RT carried out.
|
| Scan protocol |
Slides were scanned with an Axon GenePix 4000 B scanner at a resolution of 10 µm adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. Spot intensities were quantified using Axon GenePix Pro 6.0 image analysis software. The mean of the signals and the median of backgrounds were used for further analysis.
|
| Description |
Biological replicate 1 of 3
|
| Data processing |
Raw data were imported into the R 2.2.1 software (http://www.R-project.org). Background correction was done using the method substract whereas normalization of the signal intensities within slides was carried out using the printtiploess method (Yang et al., 2002) using the LIMMA package (Smyth et al., 2003, www.bioconductor.org).
|
| |
|
| Submission date |
Feb 27, 2012 |
| Last update date |
Apr 25, 2012 |
| Contact name |
Enrique Ibarra Laclette |
| E-mail(s) |
[email protected]
|
| Phone |
(462) 166 3000
|
| Organization name |
Laboratorio Nacional de Genomíca (LANGEBIO), Centro de Investigación y Estudios Avanzados del IPN (CINVESTAV)
|
| Street address |
Km 9.6 Libramiento Norte Carretera Irapuato-León, A.P. 629
|
| City |
Irapuato |
| State/province |
Guanajuato |
| ZIP/Postal code |
36821 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL7725 |
| Series (1) |
| GSE36092 |
Bifunctional Activity of members of the Arabidopsis NF-YA Transcription Factor Family |
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