Following imbibation, seeds were sown into 10 cm (length) x 10 cm (width) x 9.5 cm (depth), unvented, polycarbonate culture boxes (Sigma-Aldrich, Dorset, UK). Approximately 35 seeds were sown on perforated polycarbonate discs (diameter 91 mm; thickness 5 mm) placed over 75 mL agar containing a full strength MS basal salt mix at full strength (Murashige and Skoog 1962). Roots grew into the agar, but shoots remained on the opposite side of the disc. Boxes were placed in a growth room and transferred on their polycarbonate discs to a hydroponics system situated in a Saxcil growth cabinet 18 days after germination. In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. Plants were grown in these solutions for 7 days before they were harvested (a total of 25 days after germination). Root material from twenty to thirty plants from each treatment was bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 C prior to the extraction of total RNA.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNAeasy kit protocol
Label
biotin
Label protocol
Labelled using protocol described in manual "Affymetrix GeneChip Expression Analysis Technical Manual", One-Cycle target labelling
Hybridization protocol
Biotin Labelled cRNA using "Affymetrix GeneChip Hybridization, Wash and Stain Kit", according to guidelines set out by Affymetrix, Santa Clara, CA
Scan protocol
Scanned according to guidelines set out by Affymetrix, Santa Clara, CA.
Description
Seeds were washed in 70 % (v/v) ethanol/water, rinsed in distilled water and surface sterilised using NaOCl (1 % active chlorine). Seeds were rinsed again and imbibed for 3-6 days in sterile distilled water at 4 C to break dormancy. Growth Room Hydroponics: In the hydroponics system, plants were supported on polycarbonate discs over 450 mL of aerated nutrient solution contained in a light-proof 500 mL plastic beaker. 80% 22C KH2PO4 0.25mM KOH 0.5mM MgSO4.7H2O 0.75mM CaCl2.2H2O 0.03mM FeNaEDTA 0.1mM Ca(NO3)2.4H2O 4mM H3BO3 30uM MnSO4.4H2O 10uM ZnSO4.7H2O 1uM CuSO4.5H2O 3uM Na2MoO4.2H2O 0.5uM. Plants had fully formed rosettes but no flowerng stem. Root material from twenty to thirty plants from each treatment were bulked into 1.5 mL colourless, sterile, screw-cap polypropylene tubes and snap-frozen in liquid nitrogen. Tissue samples were stored at -70 C prior to the extraction of total RNA.
Data processing
Raw data from the microarrays was normalized at probe-level using MAS5 algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system
