Computational protocol: Pro-Inflammatory CD11c+CD206+ Adipose Tissue Macrophages Are Associated With Insulin Resistance in Human Obesity

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Protocol publication

[…] RNA was prepared from cell pellets using a Picopure RNA kit (Arcturus, Oxnard, CA). DNA was prepared by proteinase K digestion and ethanol precipitation. Primer sequences are shown (supplemental Table 2). Amplification efficiencies of primer pairs were not significantly different over the concentration ranges measured. For RT-PCR, total RNA was reverse transcribed using Superscript III (Invitrogen) and amplified on an ABI Prism 7700 platform using the Sybergreen reporter (Qiagen, Valencia, CA). Gene expression relative to β-actin was determined using the comparative CT method.Gene microarray of CD11c− and CD11c+CD206− ATMs was performed using 50 ng total RNA and human WG-6 v2 bead chips (Illumina, San Diego, CA). Microarray analysis used the lumi, limma, and annotation packages of Bioconductor (). Expression data were background corrected using negative control probes followed by a variance-stabilizing transformation () and quantile normalization. Gene-wise linear models were fitted to determine differences between cell populations, taking into account subject-to-subject variability. Significant differentially expressed genes were identified using empirical Bayes moderated t tests and a false discovery rate of 5% (). Functional annotation clustering analysis () of differentially expressed genes was performed online (www.david.abcc.ncifcrf.gov/home.jsp) using default settings. […]

Pipeline specifications

Software tools lumi, limma, DAVID
Application Gene expression microarray analysis
Organisms Homo sapiens, Mus musculus
Diseases Metabolic Diseases, Neoplasms
Chemicals Glucose