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Yotic section in the GeneChip Expression Analysis Technical Manual Rev. 3 from
Yotic section in the GeneChip Expression Analysis Technical Manual Rev. 3 from Affymetrix, and are available upon request. Arrays were scanned on a GeneChip Scanner 3000 (Affymetrix). Microarray data analysis Quality of the data was assessed on diagnostic plots generated from the raw, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25366685 non-processed data, as described [37]. All arrays passed these strict criteria and were included in the analyses.ConclusionIn conclusion, we present data showing the involvement of small intestine in the complex changes of lipid metabolism exerted by long term dietary intake of EPA and DHA by gene expression analysis and functional ex-vivo beta oxidation analysis. Furthermore, we show that these effects are regulated in a dose-dependent manner. In view of its large contribution to overall energy metabolism, modulation of gene expression and metabolism in the intestine by dietary lipids, and especially long-chain n-3 PUFA of marine origin, represents a promising target for the prevention of obesity and Cyclopamine associated co-morbidities.MethodsAnimals and diets In the first experiment, male 4-month-old C57BL/6J mice were maintained for 4 weeks on semisynthetic high-fat (20 wt/wt) diets differing in the composition of n-3 PUFA. These mice were already used in our previous study [8]. Two isocaloric diets [8,22] were used (n = 12): control sHFf diet which contained flax-seed oil (rich in ALA) as the only lipid source, or the sHFf-F2 diet, which had the same composition except that 44 of the lipids were replaced by a n-3 PUFA concentrate containing 6 EPA and 51 DHA (EPAX 1050TG; EPAX AS, Lysaker, Norway). This diet is denoted as EPA DHA throughout the study. At the end of the experiment, mice were killed by cervical dislocation and small intestine from 3 cm under the stomach to caecum was isolated and cut lengthwise. The intestine was washed in 154 mM KCl and scraped. The epithelial cells were collected, frozen in liquid nitrogen and stored at -80 . This procedure was also performed for 5 cm of colon.In the second PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24141786 experiment, subgroups (n = 9) of male 4month-old C57BL/6J mice were fed either (i) control obesity-promoting high-fat (35 lipids wt/wt; cHF) diet derived from standard chow diet based on corn oil, or (ii) and (iii) cHF diet with 15 and 44 of lipids, respectively, replaced by EPAX 1050TG (cHF-F1 and cHF-F2, respectively; see [22]). After 6 weeks, intestinal tissue was isolated for fatty acid beta oxidation and gene expressionThe Affymetrix default algorithm (MAS 5.0) was used to summarize data and significance of observed gene expression changes (present/absent call per probeset per array, fold change (FC) between normalized arrays, and its significance by the p-value). In total, 24270 probesets (54 )Page 8 of(page number not for citation purposes)BMC Genomics 2009, 10:http://www.biomedcentral.com/1471-2164/10/showed expression at least in either one of the arrays. Significant differentially expressed probesets were identified by direct comparison between the two dietary groups for all probesets called present on both arrays. Probesets that satisfied the general Affymetrix criterion of t-test probability < 0.27 (p-value < 0.0027) were considered to be significantly regulated, and these were further investigated. Probesets were annotated using information provided by Affymetrix (release of July 12th, 2006) and all gene symbols are presented throughout the article according to Mouse Genome Informatics [38]. Pathway analysis, including ranking, was done usi.
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