|
| Status |
Public on Nov 09, 2020 |
| Title |
Mouse Islet 2 [KL28846_gene-level] |
| Sample type |
RNA |
| |
|
| Source name |
KO Control Diet
|
| Organism |
Mus musculus |
| Characteristics |
age: 6 months
Sex: male
genotype: Gaz KO
diet: Control
cell type: Pancreatic islets
|
| Growth protocol |
Pancreatic islets were isolated from 37-40-week-old experimental mice (WT or GazKO; control-or HFD-fed; N=5 per group) using a collagenase digestion procedure previously described4. A stereomicroscope was used to hand-pick islets from contaminating acinar tissue into a microfuge tubecontaining 1 ml phosphate-buffered saline (PBS) and washed one additional time in PBS, with pulse centrifugation used to pellet the islets.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The PBS was aspirated, and 200 μl RLT buffer (Qiagen) added. Islets were lysed using a plastic micropestle homogenizer and RNA purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s directions. RNA quality was assessed by electrophoresis using a Eukaryote Total RNA Pico cartridge and Bioanalyzer 2100 instrument (Agilent). The minimum RNA Integrity Score (RIN) of samples subject to microarray analysis was 7.40, with most samples having RIN scores of 8.0 or above.
|
| Label |
biotin
|
| Label protocol |
Ambion GeneChip WT Expression kit ( Lot#4440536) used for labeling. Targeted 200ng RNA in-put for each sample. All protocols and procedures followed as outlined in the Ambion WT Expression Kit manual (Manual P/N 4425209 Rev. D ). All samples carried through to 16 hour IVT O/N reaction. Upon completion of IVT reactions, samples cleaned up according to the protocol and procedures outlined in the Ambion WT Expression manual and quantified on the NanoDrop Ten ug cRNA used for template input for round 2 of ss cDNA synthesis Round two ss cDNA samples purified according to the protocol and procedures outlined in the Ambion WT manual and quantified on the NanoDrop. Samples set up for fragmentation with a 5.5ug template in-put. Samples fragmented with a 5.5ug template in-put, upon completion of fragmentation 45ul transferred to new 0.2ml tube for end-terminus labeling, the remaining vol. to be used for Agilent Fragmented cDNA samples stored at -20°C 1ul used for analysis on the Agilent RNA Nano chip – tested samples look similar and within the recommended nt range All samples end-terminus labeled.
|
| |
|
| Hybridization protocol |
Sample set 01 hybridized to Mouse Exon 1.0 ST arrays at 45°C for 16 hrs O/N following all procedures outlined in the GeneChip WT Terminal Labeling and Hybridization User Manual ( P/N 702808 Rev.6) for Mouse Exon 1.0 ST Arrays
|
| Scan protocol |
GeneChips post processed on the AFX Fluidics 450 Station according to all AFX protocols and procedures defined for the Mouse Exon 1.0 ST Array (FS450 _0001) as outlined GeneChip Expression Wash,Stain and Scan user Manual (P/N 702731 rev.3) GC scanned on the GC3000 G7 Scanner (S/N 50205130).
|
| Data processing |
Raw probe cell intesity data was analyzed at both the gene- and exon-level using the Robust Multichip Analysis (RMA) algorithm within the Affymetrix Expression Console 1.3 software package. Data extracted and processed using Affymetrix Command Console version 3.2.4.1515W.
|
| |
|
| Submission date |
Jul 13, 2020 |
| Last update date |
Nov 09, 2020 |
| Contact name |
Cara Leanne Green |
| E-mail(s) |
[email protected]
|
| Phone |
6082363548
|
| Organization name |
University of Wisconsin-Madisin
|
| Department |
Medicine
|
| Street address |
2214 Kendall Ave
|
| City |
Madison |
| State/province |
Wisconsin |
| ZIP/Postal code |
53726 |
| Country |
USA |
| |
|
| Platform ID |
GPL6096 |
| Series (1) |
| GSE154325 |
Exon and Gene Level Expression Profiling of Islets in Gaz Knockout Mice on Control and High Fat Diets |
|
| Relations |
| Reanalysis of |
GSM4668998 |
