16 mice were chosen for microarray analysis from the upper and lower tails (8 each) for plasma triglyceride distribution of all the male F2 mice, excluding overtly diabetic mice. Four tissues were dissected from each mouse, producing the 64 samples.
Growth protocol
Mice were maintained on standard rodent chow with 4% fat [Harlan Teklad Rodent Diet (W) 8604, Madison, WI] ad libitum with free access to water (HCl acidified, pH 2.8-3.2) under controlled temperature and humidity with a 12-hour light and dark cycle. All animal studies were carried out with the approvals of The University of Tennessee Animal Care and Use Committee and Marshall University Animal Care and Use Committee. Mice were euthanized at 24 weeks of age by CO2 asphyxiation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from liver, muscle (combined soleus and gastrocnemius), pancreas, and adipose tissue (combined inguinal, epididymal, retroperitoneal, perirenal, and subscapular fat pads) using RNeasy Lipid Tissue Midi Kit (75842, QIAGEN, Valencia, CA) according to the manufacturer's instructions. For adipose tissue, muscle and pancreas, the entire tissue was homogenized and total RNA extracted, whereas approximately 50% of the liver was homogenized. Total RNA was further purified using RNeasy MinElute Cleanup Kit (74204, QIAGEN) for microarray analysis.
Label
biotin
Label protocol
cDNA formation and biotin labeling was done using standard Affymetrix protocols.
Hybridization protocol
Hybridizations were performed at the University of Tennessee Affymetrix Facility (Knoxville, TN) using Affymetrix GeneChip® Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) following the standard protocol.
Scan protocol
Scanning of arrays was done with an Affymetrix Genechip scanner.
Description
Mouse 639
Data processing
Individual probe data were extracted using Bioconductor (www.bioconductor.org), and gcRMA was used to produce a signal measure for each gene. Statistical analysis was performed using SAS software (Cary, NC). A mixed ANOVA model was run on the normalized data, fitting genotype and tissue treatment effects, and using array variation as the experimental error. Genes with significant (P<0.05) ANOVA interaction, and significant pair-wise False Discovery Rate were considered differentially expressed.
