Germination in a phytochamber at 26°C, with a 16 hours light, 8 hours dark cycle and 60% humidity in twice distilled water.
Extracted molecule
polyA RNA
Extraction protocol
For each biological replicate approximately 20 roots per germination roll and genotype were pooled and homogenized using the Micro-Dismembrator U (Sartorius AG, Göttingen, Germany). Isolation of total RNA with Trizol (Invitrogen, Carlsbad, CA, USA), followed by mRNA purification using Oligotex mRNA columns (Qiagen, Hilden, Germany) was performed according to the manufacturers´ instructions.
Label
Cy3
Label protocol
Reverse transcription of the mRNA to cDNA with concurrent incorporation of aminoallyl dUTPs as well as Cy3 and Cy5 labeling was performed according to Nakazono et al. (2003).
Hybridization protocol
Microarray probe hybridization of spotted 12k maize cDNA microarray chips (GenII vB, GPL 1996; www.plantgenomics.iastate.edu/maizechip) representing 10,649 non-redundant gene fragments was conducted as described in Woll et al. (2005).
Scan protocol
Dried slides were scanned six times with an Array Scanner (Genetic MicroSystems, USA) for each channel (Cy3 and Cy5) with laser power settings fixed at 90%. A series of up to six scans for each channel, in ascending order of PMT gain, was performed at 10 mm resolution individually adjusted to overall spot intensities per array according to Piepho et al. (2006). ImaGene software (Biodiscovery, Marina Del Rey, CA, USA) was used to quantify the spot intensities on the slides by the use of the default settings. ImaGene software (Biodiscovery, Marina Del Rey, CA, USA) was used to quantify the spot intensities on the slides by the use of the default settings.
Description
slide 3 3_Cy3_005x005
Data processing
The experimental design for the microarray experiments was described in Keller et al. (2005). To combine data from different scanning intensities, a nonlinear latent regression model was applied (Piepho et al., 2006). After combining the signals of different scans, a loess regression was performed. To account for differences between arrays the median absolute deviations (MAD) of the slides were normalized. The normalized data was analysed by a linear mixed model for every gene. Effects that occur during hybridization (genotype, dye, slide) were considered as well as temporal and spatial effects that might affect the genetic material during cultivation in the phytochamber (Keller et al., 2005). The unbalancedness of the design was accounted for by the mixed model analysis. Pairwise contrasts between genotypes were estimated. Furthermore, linear contrasts between hybrid and parental mean were determined. The p-values of these contrasts based on Wald tests were adjusted for multiple testing by controlling the false discovery rate (FDR) at 5% using the procedure of Benjamini and Hochberg (1995). The analyses for this paper were generated using SAS software, Version 9. Linear model analyses were done by PROC MIXED, while the FDR adjustment was performed by PROC MULTTEST.
