|
| Status |
Public on Oct 01, 2010 |
| Title |
Comparison of inoculated NIL seeds replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Eyl25
|
| Organism |
Zea mays |
| Characteristics |
near isogenic lines: Eyl25
infection: Aspergillus flavus-AF13
|
| Treatment protocol |
Seeds were surface sterilized and inoculated with A. flavus (AF13) according to the Kernel Screening Assay developed by Robert Brown. Inoculated and noninoculated seeds were incubated at 31°C and 100% humidity for 72 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from seeds using a Trizol (Invitrogen). RNA was then treated with RNase-free DNase I (Qiagen) according to the manufacturer’s instructions. Treated RNA samples were further purified using an RNAeasy clean up kit (Qiagen) to remove any residual impurities remaining.
|
| Label |
Cy5
|
| Label protocol |
Purified total RNA was amplified and labeled following the aminoallyl method (http://www.maizearray.org/maize_protocols.shtml). The Ambion Message Amp II kit was used to produce cRNA which is indirectly labeled using dye Cy3 or Cy5 coupling reaction.
|
| |
|
| Channel 2 |
| Source name |
Eyl31
|
| Organism |
Zea mays |
| Characteristics |
near isogenic lines: Eyl31
infection: Aspergillus flavus-AF13
|
| Treatment protocol |
Seeds were surface sterilized and inoculated with A. flavus (AF13) according to the Kernel Screening Assay developed by Robert Brown. Inoculated and noninoculated seeds were incubated at 31°C and 100% humidity for 72 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from seeds using a Trizol (Invitrogen). RNA was then treated with RNase-free DNase I (Qiagen) according to the manufacturer’s instructions. Treated RNA samples were further purified using an RNAeasy clean up kit (Qiagen) to remove any residual impurities remaining.
|
| Label |
Cy3
|
| Label protocol |
Purified total RNA was amplified and labeled following the aminoallyl method (http://www.maizearray.org/maize_protocols.shtml). The Ambion Message Amp II kit was used to produce cRNA which is indirectly labeled using dye Cy3 or Cy5 coupling reaction.
|
| |
|
| |
| Hybridization protocol |
According to the protocols provided by the manufacturer (http://www.maizearray.org/maize_protocols.shtml), 12 μg of Cy3 and Cy5 labeled cRNAs were hybridized to the 46k oligonucleotide array (Version 1).
|
| Scan protocol |
Slides were scanned using the Genepix 4000B (Molecular Devices, Sunnyvale, CA). The saturated spots ratio was set as 0.005%.
|
| Description |
Eyl25 Inoculated/Eyl31 Inoculated rep2
Technical replicate 2 of 4.
|
| Data processing |
Array images were processed using GENEPIX 6.0. Raw intensity data were imported into GeneSpring GX 10.0 software (Silicon Genetics, Redwood City, CA). Two criteria were used for selecting positive spots, (Signal – Background) mean > 400 unit as expression intensity filter, and at least two spots existing in the four replicates. Data normalization was performed using a LOWESS filter (locally weighted regression). Differentially expressed genes were identified by performing a one-way ANOVA on the normalized data using a T-test with no assumption of equal variance. A Benjamini and Hochberg correction for multiple testing was applied using a false-discovery rate (FDR) of 0.05. Genes showing a ratio above 2 (below 0.5) in at least three of four replicates of the same experiments were considered up- (down) regulated.
|
| |
|
| Submission date |
Jan 13, 2010 |
| Last update date |
Jan 14, 2010 |
| Contact name |
Meng Luo |
| E-mail(s) |
[email protected]
|
| Phone |
504-2864345
|
| Fax |
504-2864419
|
| Organization name |
SRRC, USDA-ARS
|
| Department |
FFS
|
| Street address |
1100 Robert E Lee Blvd
|
| City |
New Orleans |
| State/province |
LA |
| ZIP/Postal code |
70124 |
| Country |
USA |
| |
|
| Platform ID |
GPL6438 |
| Series (1) |
| GSE19883 |
Transcriptional profiles uncover Aspergillus flavus induced resistance in mature maize kernels |
|
