tissue: maize kernels name: DKC 78-15 Bt common name: MON 810 trait: Resistance to European corn borer (Ostrinia nubilalis). growing year: 2004 location: Lichtenburg input: high input
Treatment protocol
Maize kernels from all samples were couriered to Technische Universität München, Germany for milling and distribution. The maize kernels were milled using a cyclone mill (Cyclotec, Foss, Germany) equipped with a 500-µm sieve and freeze-dried (ALPHA 1-4 LSC, Christ, Osterode, Germany) for 48 hours. Aliquots of maize powders (2g) were prepared and couriered to the different laboratories for specific analysis. Upon arrival the maize powders were kept at -20 degrees Celcius and used as needed.
Growth protocol
The Bt F1 hybrids (DKC 78-15Bt), the RR F1 hybrid (DKC 78-35RR) and the isoline (CRN 3505) were grown in 2 different Monsanto sites, namely, Petit and Lichtenburg (South Africa) under high-input system; these lines were planted in Petit over three growing seasons (2004, 2005 and 2006) and in Lichtenburg over one growing season (2004). At planting the plants were fertilized with 300kg/Ha 4:3:4 (33), Topdressing 300kg/Ha KAN (28) and treated with herbicide 1.8L/Ha Guardian + 200M1/Ha Sumi Alpha. Two months after planting the plants were treated with herbicide, 2.2L/Ha A-maizing + 1L/Ha Harness + 220Ml/Ha alphacypermytrin. Three months after planting the material was treated with pesticide, 750Ml/Ha Endosulfan against stalkborer. The plant material was harvested 8 months after planting; the kernels were removed from the cobs on site by machine and packed in plastic bags. The moisture content was 11-13%. The Bt F1 hybrid (DKC 78-15Bt) and the control conventional line (CRN 3505) were also grown in Potchefstroom (South Africa) under low-input system which means that no fertilizer, no fungicide and no herbicide were applied throughout the growth of the plants. The plant material was also harvested after the cobs were dry around 8 months after planting.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction following a protocol for extraction of RNA from polysaccharide rich tissues (Chang et al.,2003,Plant Molecular Biology Reporter, 11: 113–116)
Label
Cy3
Label protocol
RNA (100μg) was labeled by incorporation of Cy3-dCTP during a cDNA synthesis reaction using 21-mer oligo-dT primers according to the method described by Boeuf et al (2001) and Franssen-van Hal et al (2002). Labeled cDNA was first dissolved in MiliQ treated water (500 μl) to which later 2X hybridization buffer was added (500 μl), pre-warmed to 60°C
Hybridization protocol
The slides were prehybridized according to the protocol described by Hedge et al, (2000).The hybridization mixture was equally dispersed over the two slides. The hybridization was performed in a hybridization chamber and gasket (Agilent, Amstelveen, Netherlands). Slides were hybridized overnight at 60°C in a rotating hybridization oven in darkness (Agilent, Amstelveen, Netherlands). After hybridization the slides were washed with wash buffer 1, 2 and stabilization and drying solution, according to the manufacturer’s instructions (Agilent, Amstelveen, Netherlands). The slides were stored at room temperature in darkness until scanning.
Scan protocol
Microarrays were scanned after excitation of Cy3 dye with 543 nm laser using scanner ScanArray® Express HT (Perkin Elmer, USA). The microarrays were scanned at constant laser power (90 %) and 10 μm resolution settings.Tiff images were imported into the ArrayVision software (Imaging Research, Waalwijk, The Netherlands) and the fluorescent intensity and background were determined for each spot.
Description
none
Data processing
Quality checked visually; control spots removed; Filtering of rows of spots with 17 out of 18 samples having a value lower than 2X background, Median normalized (array-wise, separately for the two platforms).
