|
| Status |
Public on Jan 01, 2010 |
| Title |
MO-A.1.13.261 (Cy5) |
| Sample type |
RNA |
| |
|
| Source name |
total RNA leaves
|
| Organism |
Zea mays |
| Characteristics |
treatment: 0 days stress
replicate: Biological 2
landrace: 85-2
|
| Treatment protocol |
30 day old plants were subjected to a progressive water deficit by leaving them unwatered for 10 days, 17 days (severe stress) and then given a recovery irrigation
|
| Growth protocol |
Zea mays L. seeds from 85-2, Cajete criollo and Michoacan 21 landraces were surface sterilized with NaOCl (10%) for 30 min and then washed with distilled water before sowing. After washes in distilled water, seeds were germinated and grown in 15 L plastic pots in a substrate of 92.46% sand and 7.44% clay under greenhouse conditions. Temperatures were between 19° C to 32°C and relative humidity was 60±5%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions and a second re-extraction with Plant RNA purification Reagent Concert (Invitrogen) according to the manufacturer's guidelines.
|
| Label |
Cy5
|
| Label protocol |
30 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. After hybridization, the slides were washed once in solutions 1–4 (wash solution 1: 2% SSC, 0.1% SDS at 45 ºC; wash solution 2: 0.1% SSC, 0.1% SDS; wash solution 3: 0.1% SSC; wash solution 4: 0.01% SSC) for 5 min with gentle stirring/agitation. Washed slides were dried by centrifugation at 1600 r.p.m.
|
| 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 |
none
|
| 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 and the Aquantile normalization (Yang et al., 2002) using the LIMMA package (Smyth et al., 2003, www.bioconductor.org).
|
| |
|
| Submission date |
Feb 05, 2009 |
| Last update date |
Feb 10, 2009 |
| Contact name |
June Simpson-Williamson |
| E-mail(s) |
jsimpson@ira.cinvestav.mx
|
| Phone |
(0052) 462 6239600
|
| Organization name |
Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional
|
| Department |
Plant Genetic Engineering
|
| Lab |
Plant Molecular Genetics
|
| Street address |
Km 9.6 Libramiento Norte Carretera Irapuato-Leon
|
| City |
Irapuato |
| State/province |
Guanajuato |
| ZIP/Postal code |
36821 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL8162 |
| Series (1) |
| GSE14728 |
Mexican maize landraces under drought stress and recovery irrigation |
|
