|Application:||Gene expression microarray analysis|
|Number of samples:||12|
|Release date:||Jun 30 2009|
|Last update date:||May 1 2012|
|Dataset link||Transcriptomic analysis of the effect of silicon on wheat plants infected or uninfected with powdery mildew|
Wheat culture in hydroponic system : Hydroponic systems were used to precisely control Si feeding of plants and to reduce external Si contamination. Seeds of wheat cultivar AC Drummond, chosen for its known high susceptibility to Bgt, were sown in 9-cm pots in a nylon bed consisting of nylon stockings cut into small ribbons. Hydroponic systems were set up to immerse roots for 15 minutes every 30 minutes. The plants were grown in a greenhouse (16 h light at 22°C and 8h dark at 18°C, 80% humidity). Plants were immersed in distilled water only during the first week following sowing. Si amendment and Bgt inoculation: Wheat plants were grown in the presence (Si+) or absence (Si-) of Si and inoculated (B+) or not (B-) with Bgt for a total of four treatments: Si-B-, Si+B-, Si-B+, and Si+B+. These treatments were applied in the following manner. One week after sowing, distilled water was replaced by a Hoagland solution amended or not with potassium silicate (Kasil 6, PQ Corp etc) at a concentration of 1.7 mM (Si+ Hoagland). Twice a week, Si was added to the Si+ nutrient solution in order to maintain Si concentration at 1.7 mM. Every other week, both Si- and Si+ Hoagland solutions were renewed and the pH adjusted to 5.8 in all systems at each Si addition or nutrient solution renewal. Four weeks after sowing (i.e. three weeks after Si amendment started), half the plants were inoculated with Bgt as described previously. Briefly, one day prior to inoculation, reservoir wheat plants heavily infected with Bgt were gently shaken to remove old spores and to stimulate the production of fresh ones. The inoculation was performed by shaking these infected wheat plants over the experimental plants . RNA preparation: Total RNA was extracted from leaves of three plants per treatment (three biological replicates) with the RNeasy plant kit (Qiagen, Hilden, Germany). Concentration and quality of RNA were assessed on a Nanodrop spectrophotometer (Nanodrop, Wilmington, De, USA) and 2100 Bioanalyzer (Agilent, Palo Alto, Ca, USA). Biotin-labeled cRNAs were synthesized using MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, Tx, USA) following the manufacturer’s instructions using 1 µg total RNA. Labelled cRNA was then fragmented using 5X Array Fragmentation Buffer supplied with the MessageAmp II-Biotin Enhanced Kit. Microarray hybridization: The labeled samples were added to an Affymetrix GeneChip® Wheat Genome Array (Affymetrix, Santa Clara, CA, USA), according to the manufacturer’s instructions. The wheat chip contains probe sets representing 55,052 transcripts for all 42 chromosomes in the wheat genome. The chips were hybridized for 16 h at 45 °C in a rotisserie oven at 60 rpm. Following hybridization, the arrays were washed and stained in an Affymetrix Fluidics Station 450, according to the standard protocol from Affymetrix, and scanned using an Affymetrix Scanner 3000. Twelve chips were prepared and corresponded to three biological replications of each of the four treatments (Si-B-, Si+B-, Si-B+, and Si+B+).