|
| Status |
Public on Dec 28, 2007 |
| Title |
Stage1_Vs_Stage2_13111952 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from Soybean Cotyledons in Stage 1 of germination
|
| Organism |
Glycine max |
| Characteristics |
Cultivar Williams. Pooled cotyledons from 10 seedlings in Stage 1 of germination/4 different RNA purifications pooled
|
| Biomaterial provider |
Dr. L. Vodkin’s laboratory. University of Illinois.
|
| Growth protocol |
Four Glycine max Williams dry seeds were planted per small pot (4 inches) containing pre-wetted Universal Mix SB300 soil in the green house.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
McCarty D. 1986. Phenol Chloroform, Lithium Chloride extraction method
|
| Label |
Cy3
|
| Label protocol |
Direct incorporation using Cy3-dUTP
|
| |
|
| Channel 2 |
| Source name |
Total RNA from Soybean Cotyledons in Stage 2 of germination
|
| Organism |
Glycine max |
| Characteristics |
Cultivar Williams. Pooled cotyledons from 10 seedlings in Stage 2 of germination/4 different RNA purifications pooled
|
| Biomaterial provider |
Dr. L. Vodkin’s laboratory. University of Illinois.
|
| Growth protocol |
Four Glycine max Williams dry seeds were planted per small pot (4 inches) containing pre-wetted Universal Mix SB300 soil in the green house.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
McCarty D. 1986. Phenol Chloroform, Lithium Chloride extraction method
|
| Label |
Cy5
|
| Label protocol |
Direct incorporation using Cy5-dUTP
|
| |
|
| |
| Hybridization protocol |
A total of 20 µg of the reference Stage 1 (imbibed seed) RNA was used to be labeled by reverse transcription with Cy3-dUTP and compared to 20 µg of RNA from each of stages 2-7 labeled by reverse transcription with Cy5-dUTP following the protocol by Hedge et al., 2000. The Soybean Oligo array slides were pre-hybridized, hybridized and washed as described by Vodkin et al., 2004. Duplicate dye swaps were set up as well.
|
| Scan protocol |
The hybridized slides were scanned with a ScanArray Express Microarray Scanner, Packard Bioscience/ PerkinElmer (Shelton, CT) and the fluorescence was quantified by using the ScanArray Gx/ProScanArray, Microarray Analysis System (Packard biosciences/Perkin Elmer, Shelton, CT) using a protocol defined by a corresponding GAL file previously generated.
|
| Description |
Biological replicate 1 Technical replicate 2. The quantitated GPR files were then transferred to GeneSpring 7.2 Agilent Technologies (Palo Alto,CA) for further analysis. For microarray data analysis using GeneSpring 7.2, Agilent Technologies (Palo Alto, CA) we uploaded initially a soybean Genome or Array file containing the corresponding information for each one of the Oligos represented in the array. A systematic name was defined and listed along with its corresponding EST accession number, Clone ID, Blast X (cutoff 10E-06), top hit GI number and its description, top hit Arabidopsis ID and its description, corresponding gene ontologies to top hits, and associated EST’s numbers. This file was uploaded in GeneSpring 7.2 (Silicon Genetics, CA) and was used as the soybean oligo genome information for further analysis.
|
| Data processing |
Data from a total of 48 slides, containing the intensity values for the reference and the signal channels as well as the flag values for present, absent or marginal spots was uploaded and sample attributes were defined. The data was initially transformed for dye swap for the appropriate slide samples and all the intensity values less than 0.01 or negative were converted to 0.01 to reset biologically irrelevant or otherwise unacceptable expression values. Time was defined as the only parameter to be taken into account for analysis and 6 different conditions were defined: one for each time point defined. After background subtraction and eliminating all the spots flagged as marginal or absent, data was normalized using an intensity-dependent LOWESS (locally weighted scatter plot smoothing) normalization method (Yang et al., 2002) to minimize systematic non-biological differences and standardize chips for cross-array comparisons. Normalized data was then filtered on expression level to obtain a list of genes with expression values in at least one of the comparisons made. This list was then used for statistical analysis. A Welch ANOVA (parametric test, variances not assumed equal) was carried out with a P-value cutoff 0.05, a false discovery rate (FDR) of 0.05 and between 0.1 and 100 fold expression value change. This list was further filtered on expression level creating restrictions to define relevant genes at any particular time point as well as to find expression profiles of biological relevance for the study.
|
| |
|
| Submission date |
Dec 08, 2006 |
| Last update date |
Dec 11, 2007 |
| Contact name |
Lila O. Vodkin |
| E-mail(s) |
[email protected]
|
| Phone |
217-244-6147
|
| Organization name |
University of Illinois
|
| Department |
Crop Sciences
|
| Lab |
Lila Vodkin
|
| Street address |
1201 W. Gregory Dr.
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL4635 |
| Series (1) |
| GSE6534 |
Glyoxylate pathway elements involved in the functional transition of soybean cotyledons during seedling development |
|
