|
| Status |
Public on Nov 26, 2007 |
| Title |
27_CON.I3L2.CEL, Experimental replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Soybean cultivar CONRAD - pathogen(P.sojae) Inoculated: 72 hours post inoculation : Sample taken from the lower side of the inoculation
|
| Organism |
Glycine max |
| Characteristics |
cultivar - CONRAD
Tissue/Cell Type: Soybean root/hypocotyls - P.sojae mycelium from minimal medium
total Soybean RNA
|
| Treatment protocol |
Inoculation assays were performed using the slant board technique (Olah and Schmitthenner, 1985).[ Briefly, 7-day-old soybean seedlings were thoroughly rinsed under tap water and placed in an inoculation tray (10 plants per tray, 3 trays per inoculation replication). The plants were wounded at 2 cm below the beginning of the root zone by scraping the epidermis with a scalpel and then inoculated with a mycelial slurry from a 7-day-old culture or agar alone. Samples of root tissues were collected at 3 and 5 days post inoculation (dpi) from 7.5 mm below and above the lesion margin from each seedling with characteristic lesions. For the mock-inoculated plants, tissue sections were taken 7.5 mm below and above the position corresponding to the average lesion length measured from the inoculated samples. The lesion length (mm) from the inoculation point up to the edge of the lesion margin was also measured 3 and 5 dpi. For each experimental replication, 2 replicates of 30 seedlings per condition were inoculated and harvested separately. All the plants for an experiment were grown in the same environmental growth chamber (Chagrin Falls, Ohio; Model M-48 with TC2 microcontroller unit) with day and night temperatures settings of 27C and 21C and relative humidity averaging 75 to 90%.
|
| Growth protocol |
Soybean plants were grown in growth chambers and later transferred to trays with clothes soaked in water
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAGEN RNeasy Kit : As recommended by manufacturer
|
| Label |
biotin
|
| Label protocol |
The RNA samples isolated from each of the two inoculation replications were pooled in equal amounts before subjecting to microarray assay. RNA samples from mock inoculated tissue contain 0.03125-4% spike-in RNA from Phytophthora sojae mycellium grown in liquid sucrose-salts medium , depending upon resistance level and sampling time. Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA. RNA is is first reverse transcribed using a T7-Oligo(dT) Promoter Primer in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA is purified and serves as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction is carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling. The biotinylated cRNA targets are then cleaned up, fragmented, and hybridized to GeneChip expression arrays(Affymetrix Technical manual - 2004)
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| |
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| Hybridization protocol |
hybridization cocktail is prepared, including the fragmented target, probe array controls, BSA, and herring sperm DNA. It is then hybridized to the probe array during a 16-hour incubation(Affymetrix technical manual - 2004)
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneChip Scanner 3000
|
| Description |
CONRAD - P.sojae inoculated; 72 hrs (lower); ExpRep2
Data was collected from the hypocotyl regions of Soybean plants at different time points
|
| Data processing |
The data were analyzed with RMA suite from R package version 2.4.0. Background correction was using RMA followed by quantile normalization and data was summarized using median polish method
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| |
|
| Submission date |
Nov 26, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
sucheta Tripathy |
| E-mail(s) |
sutripa@vbi.vt.edu
|
| Phone |
5402318138
|
| Organization name |
Virginia Tech
|
| Department |
Virginia Bioinformatics Institute
|
| Lab |
Tyler lab
|
| Street address |
1, Washington street
|
| City |
Blacksburg |
| State/province |
VA |
| ZIP/Postal code |
24061 |
| Country |
USA |
| |
|
| Platform ID |
GPL4592 |
| Series (1) |
| GSE9687 |
Expression patterns in time and space during P.sojae infection of soybean cultivars differing in quantitative resistance |
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