Computational protocol: Human Adipose Tissue Macrophages Are Enhanced but Changed to an Anti-Inflammatory Profile in Obesity

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Protocol publication

[…] RNA was isolated using TRIzol reagent (GIBCO-BRL Life Technologies, Roskilde, Denmark), and cDNA was synthesized using random hexamer primers using the Verso cDNA Kit (Applied Biosystems). All analyses were performed simultaneously and the mRNA levels of the target genes were expressed relative to the house-keeping gene low-density lipoprotein receptor related protein-10 (LRP10). The PCR reactions were performed in duplicate using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Inc., Woburn, MA, USA) in a LightCycler 480 (Roche Applied Science) using the following protocol: one step at 95°C for 3 min, then 95°C for 10 s, 60°C for 20 s, and 72°C for 10 s. The increase in fluorescence was measured in real time during the extension step. The relative gene expression was estimated using the default “Advanced Relative Quantification” mode of the software version LCS 480 1.5.0.39 (Roche Applied Science). The primers were designed using QuantPrime software []. The specificity was tested and amplification efficiency determined (between 1.9 and 2.0). Before analysis of target genes, the house-keeping gene was tested for stability and found to be stable comparing both groups and displaying comparable number of C T cycles. The primer pairs are listed in . [...] Descriptive statistics for anthropometrics and HOMA-IR are presented as mean ± SD. Baseline unpaired data were analyzed with an unpaired t-test or a Wilcoxon Mann-Whitney rank sum test for those variables, which were not normally distributed. To analyze the association between the cytokines, HOMA-IR, and the different macrophage markers we used a Spearman's correlation test. Multivariate linear regression was performed on a log scale to adjust the total number of macrophages and to find the macrophage marker with the closest association to HOMA-IR. The chosen significance level was a two-tailed P value of <0.05. The statistical software packet SPSS (SPSS, Chicago, IL, USA) was used for all calculations. Graphs were made in SigmaPlot. […]

Pipeline specifications

Software tools QuantPrime, SPSS, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Neoplasms, Adipose Tissue