strain: P6497, race 2 specimen: mycelia growth protocol: vegetable juice V8 media
Growth protocol
vegetable juice V8 media
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with Trizol reagent.
Label
biotin
Label protocol
Biotinylated complimentary RNA (cRNA) was prepared from 10 μg of total RNA as per the Ambion MessageAmp II protocol (Ambion, Austin, TX). Double-stranded cDNA was synthesized using ArrayScript and oligo(dT) primers with a T7 promoter. Biotin-labeled cRNA was prepared by cDNA in vitro transcription using MEGAscript. External spike-in controls (Affymetrix, Santa Clara, CA) were used.
Hybridization protocol
A cocktail containing 10 μg of labeled cRNA was hybridized to the Soybean GeneChips for 16 hours at 45°C as described in the Affymetrix Technical Analysis Manual (Affymetrix, Santa Clara, CA). GeneChips were stained with Streptavidin-Phycoerythrin, followed by an anti-streptavidin antibody solution and a second Streptavidin-Phycoerythrin solution, with all liquid handling performed by a GeneChip Fluidics Station 450.
Scan protocol
GeneChips were scanned with the Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Description
P. sojae was grown on vegetable juice (V8) agar medium containing 2.65g calcium carbonate and 260mL of V8 juice per liter. Solid media was overlaid with sterile cellophane disks prior to culture of P. sojae, for ease of removal of mycelia at sampling. Total RNA was extracted with Trizol reagent.
Data processing
Probe level signal intensities (.cel files) were generated using GCOS1.4 (Affymetrix Inc., Santa Clara, CA). Gene level data (.chp files) were generated using MAS5 default values for the Statistical Expression algorithm parameters and a Target Signal of 150 for all probe sets and a Normalization Value of 1.
