Genotype: Mo17 Tissue: above ground seedling Dev stage: 14 days old Sample name: 1686 Mo17 Cy3
Extracted molecule
polyA RNA
Extraction protocol
total RNA extraction with Trizol (Invitrogen, Carlsbad, CA) with minor modifications to manufacturer’s instructions, mRNA isolation with Oligotex Kit (Qiagen, Valencia, CA) as per manufacturer’s instructions
Label
Cy3
Label protocol
Fluorescent cDNA targets were synthesized and hybridized as described at http://schnablelab.plantgenomics.iastate.edu/resources/protocols/. Labeled targets containing a minimum of 3000 picomoles of cDNA, 50 picomoles of Cy dye, and more than one dy molecule per 50 (100) bases of Cy3 (Cy5) were used for hybridizations.
Genotype: B73 Tissue: above ground seedling Dev stage: 14 days old Sample name: 1686 B73 Cy5
Extracted molecule
polyA RNA
Extraction protocol
total RNA extraction with Trizol (Invitrogen, Carlsbad, CA) with minor modifications to manufacturer’s instructions, mRNA isolation with Oligotex Kit (Qiagen, Valencia, CA) as per manufacturer’s instructions
Label
Cy5
Label protocol
Fluorescent cDNA targets were synthesized and hybridized as described at http://schnablelab.plantgenomics.iastate.edu/resources/protocols/. Labeled targets containing a minimum of 3000 picomoles of cDNA, 50 picomoles of Cy dye, and more than one dy molecule per 50 (100) bases of Cy3 (Cy5) were used for hybridizations.
Each microarray chip (10 total) was scanned a minimum of six times in ascending amounts of laser power and PMT gain settings using a ProScanArray scanner (PerkinElmer, Boston, MA). Two scans from each chip were selected based on the median value of the natural log of the signal median for all spots on the slide.
Description
Maize seedlings were grown under controlled conditions and were harvested at the 14 day old stage. Total RNA was isolated from ten replications of B73, Mo17 maize seedling genotypes (6 individual seedlings pooled for each genotype in each replication) using Trizol reagent (Invitrogen, Carlbad, CA) with slight modifications to manufacturer’s protocol. Approximately 500 micrograms of total RNA were used for mRNA isolation with the OligoTex mRNA midi kit (Qiagen, Valencia, CA ) as per manufacturer’s protocol with slight modification. Two micrograms of mRNA were labeled according to Nakozono et al. (2003) with slight modifications. Each sample was labeled with Cy dye and hybridized to the GP2613 platform.
Data processing
The lowess normalization method of Dudoit, Yang, Callow, and Speed (2002) was applied to the log of background-corrected raw signal intensities to remove signal-intensity-dependent dye effects from each slide. Normalization was carried out separately for each slide to avoid introducing dependencies among biological replications. Following lowess normalization, the normalized data for each slide/dye combination were median centered so that expression measures would be comparable across slides. Median-centering involves subtracting the median value for a particular slide/dye combination from each individual value associated with the particular slide/dye combination. The lowess normalized data from each scan was used for a mixed linear model analysis (Wolfinger et al., 2001).
