|
| Status |
Public on Jul 02, 2008 |
| Title |
Mudanjiang8 OsWRKY13 Overexpressing D11UM7-2 rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mudanjiang8 OsWRKY13 Overexpressing tansgenic plant D11UM7-2
|
| Organism |
Oryza sativa |
| Characteristics |
OsWRKY13-overexpressing independent transgenic line T3 D11UM7-2
|
| Growth protocol |
Leaves of a 20 different plants pool were harvested from the 4-week old wild-type Mudanjiang 8 (Oryza sativa ssp. japonica) and OsWRKY13-overexpressing independent transgenic lines (D11UM1-1 and D11UM7-2) growing in the greenhouse at 26°C with 12 hours daylight. The experiment was repeated three times.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIZOL reagent (Invitrogen, Gaithersburg, MD) and was further purified with Nucleospin RNA Clean-up Kit (Macherey-Nagel, Germany) according to the manufacture’s instructions. The RNA quality was assessed by formaldehyde agarose gel electrophoresis and concentration was quantitated by spectrophotometric analysis.
|
| Label |
biotin
|
| Label protocol |
Biotin labeled cRNA was produced with MessageAmp II-Biotin Kit (Ambion Inc., Texas) with 5 ug of total RNA as starting material for each sample reaction.
|
| |
|
| Hybridization protocol |
Fifteen micrograms of the resulting biotin-tagged cRNA was fragmented to strands of 35 to 200 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual).Subsequently, 10 ug of this fragmented target cRNA was hybridized at 45°C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 320) to probe sets on an Affymetrix Genechip Rice Genome Array.
|
| Scan protocol |
The chip was washed and then stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 400, followed by scanning on a GeneChip Scanner 3000 (Affymetrix, Inc.).
|
| Description |
Microsoft Excel program was used to merge the overlapping significant genes from two transgenic generations. Annotations from Affymetrix for significant genes were checked in NCBI public database using Blastx program (Altschul et al., 1997) and in TIGR Rice Genome Annotation version 4.0 (http://rice.tigr.org, Yuan et al., 2005) and corrected if necessary.
|
| Data processing |
The results were quantified and normalized using MicroArray Suite 5.0 (GCOS1.4) software (Affymetrix, Inc.).The genes with probe set signal lower than 100 on the repeated six arrays were considered as non-expression and filtered out (Hu and Ma, 2006), resulting in 24283 expressed genes in rice line D11UM1-1 and 24293 expressed genes in D11UM7-2 for further analysis. The Significance Analysis of Microarrays (SAM) Excel Add-in (Tusher et al., 2001) was used to identify significantly differentially expressed genes between the control and OsWRKY13-overexpressing plants. The number of permutation for SAM analysis was 100. The delta value in the SAM was adjusted so that the estimated false discovery rate (FDR) was < 5% for significant genes. The cutoff value of expression fold change was set as 2.0 that was commonly used for microarray analysis to further increase the stringency (Hu and Ma, 2006).
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| |
|
| Submission date |
Jul 05, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Deyun Qiu |
| E-mail(s) |
[email protected]
|
| Phone |
86-27-87281812
|
| Fax |
86-27-87287092
|
| Organization name |
Huazhong Agricultural University
|
| Department |
College of Life Science and Technology
|
| Lab |
National Center of Plant Gene Research (Wuhan)
|
| Street address |
Luoshi Road
|
| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430070 |
| Country |
China |
| |
|
| Platform ID |
GPL2025 |
| Series (1) |
| GSE8380 |
Rice Gene Network Inferred from Expression Profiling of Plants Overexpressing OsWRKY13 |
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