|
| Status |
Public on Aug 01, 2011 |
| Title |
polysomal RNA_biological replicate 3 |
| Sample type |
other |
| |
|
| Source name |
polysomal, germinated embryonic axes
|
| Organism |
Zea mays |
| Characteristics |
developmental stage: germinated
treatment: no insulin
|
| Treatment protocol |
Dissected maize embryonic axes from seeds imbibed during 22 h at 27°± 1°C in darkness were incubated in nutrient-complete MS medium (Murashige and Skoog, 1962) for an additional 2 h, in the presence or absence of 200 µU/mL of bovine insulin (Sigma-Aldrich).
|
| Growth protocol |
Maize seeds were germinated in a Griffin incubator for 24 hrs on a water moisturized cotton at 27°± 1°C. Embryonic axes were manually exceded for both the quiescent and germinated seeds and frozen in liquid nitrogen immediately.
|
| Extracted molecule |
other |
| Extraction protocol |
Total RNA was extracted from 1 g of quiescent or germinated embryonic axes using the TRIzol reagent as per manufacturers instructions. Polysomal RNA was extracted from 2 g of germinated embryonic axes after a sucrose cushion polysome concentration step. The polysomal fraction was resuspended in 500 µL of buffer B3 (200 mM Tris-HCI pH 8.4, 200 mM KCl, 25 mM EGTA, 36 mM MgC12, 5 mM DTT, 50 mg/mL cycloheximide, 25 mL of SDS 10% and 1 mL of proteinase K [1 mg/mL]), mixed and incubated at 37°C for 30 min. Then TRIzol reagent and chloroform were added. Isopropanol was added to the aqueous phase to precipitate the RNA.
|
| Label |
biotin
|
| Label protocol |
4 ug of total RNA as starting material were used for the preparation of biotinylated cRNA , according to the standard IVT Labeling Affymetrix protocol ( GeneChip Expression Analysis Technical Manual 2005-2006, Affymetrix).
|
| |
|
| Hybridization protocol |
10 micrograms of fragmented and labeled cRNA were hybridized for 16 hr at 45C to the Maize GeneChip Genome array. Microarrays were washed and stained in the Affymetrix Fluidics Station 450 with the Midi_euk2v3 protocol.
|
| Scan protocol |
Genechips were scanned using the Affymetrix GCOS software and the Affymetrix Genechip Scanner 3000 7G.
|
| Data processing |
The data was analyzed using R and Bioconductor softwares. Two normalization processes were performed: quantile normalization and Loess normalization .To identify differentially expressed genes, a linear model with the empirical Bayes approach was implemented using the Limma package (Smyth, 2004 and 2005).
|
| |
|
| Submission date |
Mar 02, 2011 |
| Last update date |
Aug 01, 2011 |
| Contact name |
Laura I Uribe |
| E-mail(s) |
[email protected]
|
| Phone |
+525553501909
|
| Organization name |
INMEGEN
|
| Department |
Research
|
| Lab |
UGAE Affymetrix
|
| Street address |
Periferico Sur 4124 Col. Ex rancho de Anzaldo
|
| City |
Mexico City |
| State/province |
DF |
| ZIP/Postal code |
01900 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL4032 |
| Series (1) |
| GSE27648 |
Expression profile of Maize (Zea mays L.) Embryonic Axes During Germination: Regulation of Ribosomal Protein mRNAs. |
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