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| Status |
Public on Jul 19, 2010 |
| Title |
P. sojae strain P6954 - race 1: Treated - 3 Hours post inoculation - rep 3 |
| Sample type |
RNA |
| |
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| Source name |
P.sojae strain P6954 - race 1: Treated - 3 Hours post inoculation
|
| Organism |
Phytophthora sojae |
| Characteristics |
strain: P6954
time: 3 h
|
| Treatment protocol |
Agar plugs of P. sojae isolate P6954 (race 1) were transferred to Petri dishes containing V8 liquid medium and incubated at 25C. P6954 is genetically near-identical to P6947 (Forster et al, 1994) but unlike P6947, P6954 expresses Avr1b. After 2 days the mycelia were transferred to Petri dishes with water to wash excess V8 medium. Seedlings of highly-susceptible soybean cultivar Sloan were grown in the greenhouse with natural light for 9 days.After harvesting, seedling roots were washed gently to remove soil and then placed on moist cloth. Washed P. sojae mycelia were used to inoculate seedling hypocotyls: a small (~1 cm long) longitudinal slit was cut near the top half of the hypocotyls and the mycelia were placed inside the cut. After 3, 6 or 12 h, the tissue surrounding the infected hypocotyl slits. As a 0 h control, pure P6954 mycelial inocula were also harvested right after the wash step (no infection). All samples were transferred to liquid nitrogen and frozen at 80C. The entire experiment was replicated three times. .
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| Growth protocol |
P.sojae mycelia were cultured in V8 media at 25C for 2 days prior to inoculation onto Soybean hypocotyl.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAGEN RNeasy Kit : As recommended by manufacturer
|
| Label |
biotin
|
| Label protocol |
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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| 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)
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| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneChip Scanner 3000
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| Description |
Data was collected from the hypocotyl regions of Soybean plants at different time points
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| Data processing |
The data were analyzed with GCRMA suite from R package version 2.4.0. Background correction was using GCRMA followed by a modified quantile normalization routine where data from control chips are used to rank probes instead of averaging the probe values. Data was summarized using a memory efficient median polish method
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| Submission date |
Jul 15, 2010 |
| Last update date |
Jul 19, 2010 |
| 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) |
| GSE22978 |
Microarray Analysis of Phytophthora sojae Gene Expression During Early Infection |
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