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28 RNA yields were determined spectrophotometrically at 260?nm. 15??g of RNA from each sample was then denatured by incubation at 52��C in a solution of 1?M glyoxal and 50% dimethylsulfoxide in 0.1?M NaH2PO4 and separated electrophoretically on a 1% agarose gel. The quality of RNA loaded on the gel was assessed as for liver. IGF-1 mRNA levels in the liver were measured using real-time PCR. cDNA for reverse transcriptase (RT)�CPCR was synthesized using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). One microgram of total RNA was reverse transcribed using random hexamer primers and SuperScript II reverse transcriptase according to manufacturer's protocol. Real-time PCR primers that are specific for rat IGF-1 (forward: http://www.selleck.cn/products/PD-0332991.html 5'-CCGGACCAGAGACCCTTTG-3'; reverse: 5'-CCTGTGGGCTTGTTGAAGTAAAA-3') and rat 18S ribosomal RNA (18S) (forward: 5'-GGACCAGAGCGAAAGCATTTGC-3'; reverse: 5'- CGCCAGTCGGCATCGTTTATG-3') were synthesized by Operon Biotechnologies (Huntsville, AL, USA). IGF-1 and 18S real-time PCR reactions were performed using DyNAmo HS SYBR Green qPCR kit (New England Biolabs, Ipswich, MA, USA). A standard curve that was generated from serial dilutions of purified plasmid DNA that encoded the respective genes was used to measure mRNA transcript copy number. mRNA data represent normalized copy number of IGF-1 using the 18S housekeeping gene. IGF-1 mRNA levels in bone were measured by RNase protection assay, as described previously.29 The effects of treatment were analyzed using a one-way ANOVA followed by a Student-Newman-Keuls multiple-comparison test (SPSS 13.0, SPSS, Inc., http://www.selleckchem.com/products/cb-5083.html Chicago, IL, USA). When the ANOVA assumptions of normality or homogeneity of variance were not met, a Kruskal-Wallis rank test followed by Dunn's multiple-comparison test was used. Differences were considered significant at p? http://www.selleckchem.com/products/a-1155463.html (see Fig. 1F) levels in bone compared than control rats. In addition, the fatty acid profile was altered in the HYPOX animals (see Fig. 1G); compared with controls, HYPOX rats had higher levels of 16:1,N-7 and 18:2,N-6 and lower levels of 18:0. Representative photomicrographs of bone marrow from a control (Fig. 2A) and a HYPOX (Fig. 2B) rat clearly illustrate the dramatic increase in marrow adiposity following HYPOX. Compared with control rats, HYPOX rats had higher adipocyte number (see Fig. 2C) and size (see Fig. 2D), resulting in a greater percentage of marrow area occupied by adipocytes (see Fig.