se tissue samples were harvested and rapidly snap frozen in liquid nitrogen, pulverized, then stored at 80 C until analysis. Adipose samples http://www.selleckchem.com/products/crenolanib-cp-868596.html from five birds in each group were used for both microarray and metabolomic analyses. Gene expression Total RNA was isolated from chicken adipose samples using the RNeasy Lipid kit and incorporating an on column DNase treated with the RNase free DNase Set according to the manufacturers protocol. RNA quality and concentration were measured using the Experion System, only RNA passing recom mended standards of quality was used for further studies. Transcriptome profiling was performed by Genome Quebec using the Affymetrix Gene Chip Chicken Genome Array. Microarray data from this study are deposited in the Gene Expression Omnibus under the accession number GSE35581.

For real time RT PCR validation, cDNA was synthesized using the iScript cDNA Synthesis kit. Com mercially designed and validated primer sets and the associated SYBR Green master mix were used to assay gene expression on a CFX96 real time PCR detection system. All samples were analyzed in triplicate and normalized to ? tubulin. Relative differences in gene expression were determined using the 2 CT method and statistical differences were tested by analysis of variance. Liquid chromatography coupled with tandem mass spectrometry Abdominal adipose tissue samples from five birds in each treatment group were extracted by placing tissue in a mortar containing liquid nitrogen and then powdering with a pestle. Portions of the powered tissue were weighed into 1. 5 mL centrifuge tubes.

Chilled methanol and internal standard aminomethane in positive mode were added to each tube. Each tube was mixed Batimastat thor oughly by vortexing for two minutes, and the metabo lites were extracted from the tissue for 30 min at 4 C. The tubes were then centrifuged and supernatant was split into two autosampler vials. One of these samples was immediately placed on the LC MS MS for analysis, while the other was stored at ?80 C for analysis in the opposite polarity ion mode on the following day. Samples were placed in an autosampler tray chilled to 4 C, and 10 uL of each was injected onto an LC column for analysis. The chromatography method for positive ion mode was reported previously by Bajad and cowor kers, with one exception that the column was cooled to 10 C.

The chromatography method for nega tive ion mode till was performed as reported by Waters and coworkers, except the gradient was allowed to run 50 min instead of 45 min to allow more thorough equili bration of the column. The eluent was introduced dir ectly into the MS via an electrospray ionization source fitted to a Finnigan TSQ Quantum Discovery Max triple quadrupole MS through a 0. 1 mm internal diameter fused silica ca pillary. The spray voltage was 4500 V in positive mode or 3000 V in negative mode. The sheath gas was set to 40 psi, and the capillary temperature was set to 290 C. The collision cell gas was set to a pres sure of