|
| Status |
Public on Nov 20, 2009 |
| Title |
13211142 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Seedling stage above ground tissue
|
| Organism |
Zea mays |
| Characteristics |
genotype: BxRILM0317
tissue: Above ground seedling
developmental stage: 14 days old
sample name: Rep 2 BxRILM0317
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA extraction with Trizol, 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/
|
| |
|
| Channel 2 |
| Source name |
Seedling stage above ground tissue
|
| Organism |
Zea mays |
| Characteristics |
genotype: RILM0317
tissue: Above ground seedling
developmental stage: 14 days old
sample name: Rep 2 RILM0317
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA extraction with Trizol, 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 dye 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 was scanned a minimum of four times with constant PMT and increasing laser power settings. Slides were scanned with a Pro Scan Array HT (Perkin Elmer, Wellesley, MA).
|
| Description |
Maize seedlings were grown under controlled conditions and were harvested at the 14 day old stage. Total RNA was isolated from four replications of RIL@, B73xRIL, and Mo17xRIL for 30 RILs (with 6 individual seedlings pooled for each genotype in each replication) using Trizol reagent. 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 GPL3333 platform (4 replications) and GPL3538 platform (2 replications).
|
| Data processing |
All scans from each slide were quantified using ImaGene (BioDiscovery, El Segundo, CA). For each slide and dye combination, median scan intensities were extrapolated using data from the multiple scans collected using a variation of a previously published linear regression method (DUDLEY et al. 2002). Non-informative spots were removed prior to analysis. In total, 14,401 (GPL3333) and 14,260 (GPL3538) informative spots were analyzed. Within-slide correlation coefficient between the Cy3 and Cy5 background-corrected raw signal intensities was used to quantify the quality of each slide in the eQTL experiment. A correlation coefficient cutoff of 0.90 flagged 4% (N=15) of the SAM1.1 slides for removal prior to analysis. In many cases, these slides had visually apparent technical artifacts. Using the same cutoff for SAM3.0 slides, less than 1% (N=1) of the slides were below this cutoff, and none were removed. For the remaining slides, lowess normalization was applied to the natural log (ie, log base e) of the background-corrected raw signal intensities to remove signal-intensity-dependent dye effects on each slide (CUI et al. 2003; DUDOIT et al. 2002). Normalization was conducted separately for each slide to avoid introducing dependencies among expression values. After normalization, data for each slide/dye combination were median-centered so expression measures would be comparable across slides. Post-normalized data was summarized by taking the average of all technical and biological replicates within a genotype.
|
| |
|
| Submission date |
May 29, 2009 |
| Last update date |
Nov 20, 2009 |
| 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 |
GPL3333 |
| Series (1) |
| GSE16136 |
Regulation of Gene Expression and Parent-of-Origin Effects in Maize Hybrids |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized log e ratio value of background corrected intensities of red channel and green channel (ch2_norm - ch1_norm) |
| 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) |
