|
| Status |
Public on Aug 02, 2012 |
| Title |
pancreatic epithelia-e15.5-MUT-2 |
| Sample type |
RNA |
| |
|
| Source name |
pancreatic epithelia e15.5
|
| Organism |
Mus musculus |
| Characteristics |
strain: Sox9-flox [mixed FVB/N x C57Bl/6J x CD1 background] X Rosa26-CreER [C57Bl/6J background]
tissue: pancreatic epithelia
developmental stage: e15.5
genotype: Sox9 mutant
|
| Treatment protocol |
Dams were injected with 6 mg/40 g tamoxifen in corn oil at e12.5.
|
| Growth protocol |
All animal experiments described herein were approved by the University of California, Irvine and San Diego Institutional Animal Care and Use Committees. For timed matings, noon on the day of vaginal plug appearance was designated e0.5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
E15.5 whole pancreatic epithelia (including both dorsal and ventral pancreatic epithelia) were manually microdissected and stored in 350 μl of RLT lysis buffer (QIAGEN, Valencia, CA) in the –80°C freezer. Each individual RNA sample was collected from four pancreata (i.e. four embryos) as per the manufacturer's instructions. We prepared RNA samples in ~12 μl of DEPC-treated water. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Approximately 250 ng of total RNA was amplified and labeled with Cy3 using the QuickAmp Labeling Kit, One-Color (Agilent Technologies, Santa Clara, CA). This labeling reaction produces 3 – 4.5 μg of Cy3-labeled cRNA (anti-sense), by first converting mRNA primed with an oligo (d)T-T7 primer into dsDNA with MMLV-RT and then amplifying the sample using T7 RNA Polymerase in the presence of Cy3-CTP.
|
| |
|
| Hybridization protocol |
1.65 μg of cRNA was fragmented and hybridized to the array for 17 hr. at 65°C then washed.
|
| Scan protocol |
Arrays were scanned with the AgilentTechnologies Scanner G2505B US23502657using protocol version GE1-v5_95_Feb07
|
| Data processing |
Mean foreground intensities were obtained for each spot and imported into the mathematical software package “R”, which is used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White specifically for this analysis. In outline, the Cy3 (green) intensities were not background corrected (this has been shown to only introduce noise), and corrected for the scanner offset (40 was subtracted for each intensity). The dataset was filtered to remove positive control elements and any elements that had been flagged as bad. Using the negative controls on the arrays, the background threshold was determined and all values less than this value were set to the threshold value. Finally, the data was normalized using the Limma Quantile Normalization package in “R” (Smyth 2004, Bolstad et al., 2003).
In designing the interest statistic for data analysis we borrowed ideas from the software package Focus (Cole et al. 2003). The interest statistic reflects a biologists understanding that a gene with a greater fold change (in absolute value) than other genes is potentially more interesting. Also, given two genes with the same fold changes, it is the gene with a higher expression level (and therefore higher absolute change) that is more relevant. This approach is described in greater detail elsewhere (Ogawa et al. 2004)
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| |
|
| Submission date |
Apr 16, 2011 |
| Last update date |
Aug 02, 2012 |
| Contact name |
Brandon Taylor |
| Organization name |
University of California, San Diego
|
| Department |
Pediatrics and Cellular & Molecular Medicine
|
| Lab |
Maike Sander, M.D.
|
| Street address |
Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Dr., Room 3101
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE28670 |
Identification of Sox9-Regulated Pathways During the Secondary Transition Stage of Pancreas Development |
| GSE28671 |
Identification of Sox9-Regulated Pathways During Pancreas Development |
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