|
| Status |
Public on Mar 01, 2008 |
| Title |
Pancreas_P1_HNF6-Tg_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
pooled pancreata, post-natal day 1, HNF6 Transgenic
|
| Organism |
Mus musculus |
| Characteristics |
Strain: B6D2
Developmental stage: post-natal day 1 Tissue: pooled pancreata
Genotype: HNF6 (hepatic nuclear factor 6) transgenic (overexpression)
|
| Treatment protocol |
HNF6 expression was driven specifically to pancreatic endocrine cells beginning at embryonic day (e) 11.5 using a 1 kb enhancer from the 5’ pdx1 promoter region. This transgenic line, termed pdx1PBHnf6, was maintained on an inbred hybrid (B6D2) background. All mice were kept on a 12 hour light/dark cycle, fed Mouse diet 5015 (LabDiet), and given water ad lib. For embryonic analyses, the morning of the vaginal plug was considered e0.5. All mouse studies were performed in accordance with the Vanderbilt Institutional Animal Care and Use Committee guidelines under the supervision of the Division of Animal Care.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pancreata were dissected at e18.5 and postnatal day 1 in ice cold PBS and immediately placed into RNAlater (Ambion). Total pancreatic RNA was isolated using RNAqueous RNA Isolation Kit (Ambion) according to the manufacturer’s instructions. Agilent’s Bioanalyzer microfluidic assay (Agilent Technologies, Palo Alto CA) was used to assay RNA integrity. Spectrophotometric and fluorometric methods were combined to quantitate protein and nucleic acids present in the sample, and to ensure quality control of each sample. RNA samples were bioanalyzed individually and highly pure samples were pooled according to their genotype (3-5 animals per pool) in order to obtain an adequate quantity of RNA.
|
| Label |
biotin
|
| Label protocol |
Following quality control, the RNA was prepared for microarray analysis using the standard Affymetrix protocol (Affymetrix Inc, Santa Clara, CA). Briefly, a total of 5 µg of total RNA was reverse transcribed to double-stranded (ds) cDNA using an oligo-dT primer coupled to a T7 promoter. In vitro transcription from the ds cDNA was then carried out using T7 polymerase and incorporating biotin-modified CTP and UTP ribonucleotides.
|
| |
|
| Hybridization protocol |
The biotinylated cRNA (15 µg) was fragmented and hybridized to an Affymetrix GeneChip Mouse Genome 430 2.0 Array containing 45,000 sets of 11 to 25-mer oligomers, representing 39,000 mouse transcripts (34,000 are annotated as well-defined genes). Hybridized cRNA was detected using streptavidin coupled to phycoerythrin.
|
| Scan protocol |
GeneChips were scanned using GeneChip Scanner 3000 7G Plus 2 and GeneChip Operating System (GCOS, Affymetrix, Santa Clara, CA). Default values were used to grid images (.DAT) and generate .CEL and .CHP files.
|
| Description |
No additional description necessary
|
| Data processing |
CEL files (raw Affymetrix data) were imported in GeneSpring 7.0 (Agilent Technologies) and transformed by RMA (Robust Multichip Analysis). The
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| |
|
| Submission date |
May 25, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Laura Crawford |
| Organization name |
Vanderbilt University
|
| Department |
Diabetes, Endocrinology, & Metab Division
|
| Lab |
Gannon Lab
|
| Street address |
715 PRB 2220 Pierce Ave
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE4926 |
Gene expression profiling of a mouse model of islet dysmorphogenesis |
|
| Relations |
| Reanalyzed by |
GSE119085 |
