According to standard protocols, the fasting venous blood samples were collected in BD Vacutainer® blood tubes. An earlier publication provided a thorough explanation of the sample collection and biochemical analysis methods [43 (link)]. Chemical-clinical analyses were done as part of routine laboratory testing, in a central laboratory (the University of Bremen, Centre for Biomolecular Interactions Bremen-CBIB). Serum samples stored at −80 °C were used to detect levels of hs-CRP, (using either single or MULTI-SPOT® Assay Systems, Meso Scale Discovery, Rockville, MD, USA).
Taking advantage of the qPCR array technology, we previously determined c-miRNA profiles in children and adolescents of the I.Family study [40 (link)]. Methods for miRNA extraction and screening from plasma samples have been previously published [35 (link),40 (link)]. Individual plasma samples were first tested for hemoglobin levels and hemolyzed samples were omitted from the analysis [35 (link)]. Different assays were performed in triplicate by using the miScript Primer Assays according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Relative miRNA levels were normalized using the endogenous spike-in Cel-miR-39 [35 (link)] employing the Data Assist v3.1 software package (Life Technologies, Thermo Fisher Scientific, Milan, Italy).
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