|
| Status |
Public on May 14, 2014 |
| Title |
mock at blister, rep1 |
| Sample type |
RNA |
| |
|
| Source name |
maize B73 kernels, blister stage, mock inoculated
|
| Organism |
Zea mays |
| Characteristics |
line: B73
tissue: kernels
developmental stage: blister
treatment: mock inoculated
|
| Treatment protocol |
Maize kernels were mock inoculated at the blister (R2) or dough (R4) stage or inoculated with A. flavus at the blister (R2), milk (R3), dough (R4), or dent (R5) stage, and harvested 4 days later.
|
| Growth protocol |
Inbred maize genotype B73 was grown at the Central Crops Research Station in Clayton, NC. A. flavus NRRL 3357 was grown on potato dextrose agar (PDA) at 28°C for 7-10 days. Conidia were dislodged with 0.05% (v/v) Triton X-100 and diluted to a working solution of 1x10^6 spores mL-1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Eight kernels per rep were ground into a fine powder using a pestle in a mortar containing liquid nitrogen and glass beads (Sigma, St. Louis, MO). The ground kernel tissue was immediately transferred to an Oak Ridge centrifuge tube containing 5 ml phenol. The tube was vortexed, 5 ml of 2M Tris buffer solution pH 7 (Acros Organics, Geel, Belgium) was added, and the tube was vortexed again. The tubes were spun in a centrifuge at 10K rpm for 10 minutes at 4°C. The supernatant was transferred to another Oak Ridge centrifuge tube containing 5 ml of 5:1 Phenol: Chloroform at pH 4.5 (Ambion, Foster City, CA). The tubes were vortexed and spun in the centrifuge at 10K rpm for 10 minutes at 4°C. The supernatants were transferred to an additional Oak Ridge centrifuge tube and the Phenol: Chloroform extraction was repeated. The supernatant was transferred to another Oak Ridge centrifuge tube and two volumes of ice cold 95% ethanol were added. The tubes were incubated at -20ºC for 1 hour and then spun at 10K rpm for 30 minutes at 4°C. The liquid was decanted and the pellets were dried. 450 μl of buffer RLT was added as per the RNeasy RNA isolation kit instructions (Qiagen, Valencia, CA) and the rest of the protocol was performed, including DNase treatment.
|
| Label |
biotin
|
| Label protocol |
Standard Affymetrix protocol.
|
| |
|
| Hybridization protocol |
Standard Affymetrix protocols were followed for hybridization of single-stranded cRNA to the single-stranded DNA oligonucleotides on the Affymetrix GeneChip®.
|
| Scan protocol |
Standard Affymetrix protocols were followed to generate .DAT and .CEL files (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
|
| Description |
Wounded_1Blister_Rep1
Transcriptional changes in mock-infected maize kernels.
|
| Data processing |
CEL files generated from the GeneChip DNA microarray scans were imported into JMP/Genomics and log2 transformed for each maize and A. flavus probe. Mismatch probe data were discarded. Log2 intensities were normalized separately for maize and A. flavus probes using the Loess normalization procedure implemented in JMP/Genomics. QC checks were performed before and after normalization. Final expression intensities for each probe set were generated by averaging across data from individual probes. Arrays for the mock-inoculated treatment that had moderate-to-strong A. flavus signal intensities were removed from further analysis. While these kernels visually did not appear infected, they were likely inadvertently contaminated with A. flavus.
|
| |
|
| Submission date |
May 13, 2014 |
| Last update date |
Oct 29, 2014 |
| Contact name |
Gary Payne |
| Organization name |
North Carolina State University
|
| Street address |
851 Main Campus Drive
|
| City |
Raleigh |
| ZIP/Postal code |
27606 |
| Country |
USA |
| |
|
| Platform ID |
GPL8345 |
| Series (1) |
| GSE57629 |
Transcriptional changes in Aspergillus flavus-infected maize kernels |
|
