|
| Status |
Public on Dec 31, 2008 |
| Title |
B6-high glucose-3 |
| Sample type |
RNA |
| |
|
| Source name |
C57BL/6 pancreatic islets
|
| Organism |
Mus musculus |
| Characteristics |
high glucose
|
| Treatment protocol |
Islets were then cultured separately for 24 h in RPMI-1640 media containing either 5 mM or 20 mM glucose.
|
| Growth protocol |
Pancreatic islets were isolated from 12 week old mice. Prior to all experiments islets were cultured for 48 h in RPMI-1640 media containing 11 mM glucose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the end of the culture pretreatment the islets were picked from the culture vessel, rinsed briefly in HBSS/FBS buffer and homogenized in RLT buffer. RNA was prepared using the micro-RNAeasy kit.
|
| Label |
Cy3/Cy5
|
| Label protocol |
Two hundred nanograms of total RNA from each sample was separately amplified and labeled with Cy3- and Cy5-labeled CTP using the Agilent low input linear amplification kit Labeled cRNA was purified using the Qiagen RNeasy Mini kit protocol for liquid samples.
|
| |
|
| Hybridization protocol |
Purified cRNA was quantified and similar amounts of Cy3- and Cy5-labeled cRNA were combined, fragmented, hybridized, and washed following Agilent Two-Color Microarray-Based Gene Expression Analysis Protocol v4.0.2.
|
| Scan protocol |
Arrays were scanned at 10um, 100% PMT Green, 100% PMT Red on the Agilent Microarray Scanner G2505B.
|
| Description |
Agilent's Whole Mouse Genome Oligo microarray is comprised of 41,534 60-mer oligonucleotide probes representing over 41,000 mouse genes and transcripts
Cy3 data from: 060806_251269420804_S01_44k_Feature_Extraction.txt
Cy5 data from: 060806_251269420835_S01_44k_Feature_Extraction.txt
|
| Data processing |
Array data was extracted using Agilent Feature Extraction Software v8.1, and analyzed as dye normalized relative fluorescence units (RFUs.) Dye normalization was accomplished by pairing Cy-3 and Cy-5 values, and determining the effect of dye on RFU magnitude by including dye as a factor in an ANOVA analysis of the binary logarithm transformed probe intensities, including all two factor interactions. The other factors in the ANOVA were strain and glucose pretreatment. Found to be significant, the effect of reporter dye was subtracted in a least-squares manner, followed by channel averaging for each probe.
|
| |
|
| Submission date |
Apr 24, 2008 |
| Last update date |
Jan 05, 2012 |
| Contact name |
Dan Baker |
| E-mail(s) |
bakerd@amgen.com
|
| Phone |
805-447-1000
|
| Organization name |
Amgen Inc.
|
| Department |
Molecular Sciences
|
| Street address |
1 Amgen Center Drive
|
| City |
Thousand Oaks |
| State/province |
CA |
| ZIP/Postal code |
91320 |
| Country |
USA |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE11257 |
Pancreatic islet expression profiling in diabetes prone C57BLKS/J mice vs C57BL6/J mice. |
|
