Total RNA was obtained from frozen tissues, as previously described [36 (link)], using RNeasy Lipid Tissue (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Gene expression analysis levels were determined using the relative expression method with the threshold crossing point (Ct-value), as described [26 (link),30 (link),37 (link)].
PCR arrays for the RT2 Profiler™ PCR Array Mouse Adipogenesis (Qiagen, product no. 330,231 and Cat. No. PAMM-049Z) were performed following the manufacturers’ protocol. Gene expression levels were calculated using the ΔΔCt method after normalization to the housekeeping gene expression and determination of the fold change. Each GeneQuery™ plate contains eight controls, five target housekeeping genes (β-actin, GAPDH, LDHA, NONO, and PPIH), and genes encoding for pre-adipocyte cell markers, proliferation, differentiation and adipogenesis, lipid metabolism, and obesity. Gene expression is presented as the log10 of mean values (n = 3 in each group), as previously described [26 (link),40 (link),41 ] (https://CRAN.R-project.org/package=gplots , accessed on 4 March 2022).
PCR arrays for the RT2 Profiler™ PCR Array Mouse Adipogenesis (Qiagen, product no. 330,231 and Cat. No. PAMM-049Z) were performed following the manufacturers’ protocol. Gene expression levels were calculated using the ΔΔCt method after normalization to the housekeeping gene expression and determination of the fold change. Each GeneQuery™ plate contains eight controls, five target housekeeping genes (β-actin, GAPDH, LDHA, NONO, and PPIH), and genes encoding for pre-adipocyte cell markers, proliferation, differentiation and adipogenesis, lipid metabolism, and obesity. Gene expression is presented as the log10 of mean values (n = 3 in each group), as previously described [26 (link),40 (link),41 ] (
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