|
| Status |
Public on Jun 19, 2008 |
| Title |
S5_L |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Seedling stage above ground tissue
|
| Organism |
Zea mays |
| Characteristics |
Genotype: B73
Tissue: above ground seedling
Dev stage: 14 days old
Sample name: P3 B73 Cy3
|
| Extracted molecule |
total 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.
|
| |
|
| Channel 2 |
| Source name |
Seedling stage above ground tissue
|
| Organism |
Zea mays |
| Characteristics |
Genotype: F1
Tissue: above ground seedling
Dev stage: 14 days old
Sample name: P3 F1 Cy5
|
| Extracted molecule |
total 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.
|
| |
|
| |
| Hybridization protocol |
Fluorescent targets were hybridized as described in the “cDNA microarray protocol” file at http://schnablelab.plantgenomics.iastate.edu/resources/protocols/.
|
| Scan protocol |
Each microarray chip (27 total) was scanned a minimum of six times in ascending amounts of laser power and PMT gain settings. Slides 1-15 were scanned using the ScanArray5000 (Packard, Meriden, CA). Slides 16-27 were scanned with a Pro Scan Array HT (Perkin Elmer, Wellesley, 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 nine replications of B73, Mo17, and F1 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 hybridize 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).
|
| |
|
| Submission date |
Dec 01, 2005 |
| Last update date |
Jun 19, 2007 |
| Contact name |
Patrick S. Schnable |
| E-mail(s) |
schnable@iastate.edu
|
| Phone |
515-294-0975
|
| Organization name |
Iowa State University
|
| Street address |
2035B Roy J Carver Co-Lab
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011 |
| Country |
USA |
| |
|
| Platform ID |
GPL2613 |
| Series (2) |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized log ratio value of background corrected intensities of red channel and green channel |
| Ch1_Signal Mean |
Pixel intensity avaeraged over the local signal region for green channel (Cy3) |
| Ch1_Background Mean |
Pixel intensity avaeraged over the local background region for green channel (Cy3) |
| Ch1_Signal Median |
Median pixel intensity computed over the local signal region for green channel (Cy3) |
| Ch1_Background Median |
Median pixel intensity computed over the local background region for green channel (Cy3) |
| Ch2_Signal Mean |
Pixel intensity avaeraged over the local signal region for red channel (Cy5) |
| Ch2_Background Mean |
Pixel intensity avaeraged over the local background region for red channel (Cy5) |
| Ch2_Signal Median |
Median pixel intensity computed over the local signal region for red channel (Cy5) |
| Ch2_Background Median |
Median pixel intensity computed over the local background region for red channel (Cy5) |
| Ch1_norm |
Background corrected and normalized log value of green channel(Cy3) |
| Ch2_norm |
Background corrected and normalized log value of red channel(Cy5) |
