Adipocyte Total RNA Extraction and Gene Expression Analysis

Total RNA was extracted from adipocytes using TriReagent (Sigma-Aldrich). Briefly, cells were harvested, centrifuged, and 1 mL of TriReagent was added to the cell pellet. Cells were incubated for 8 min on ice; next, 200 μL of chloroform (Sigma-Aldrich) was added, and samples were mixed vigorously and centrifuged for 20 min at 4 °C. The aqueous phase was transferred to a new Eppendorf tube and RNA was precipitated with isopropanol (Sigma-Aldrich) and centrifuged at the maximum speed for 15 min at 4 °C. The RNA pellet was washed with 70% of ethanol (Chempur, Karlsruhe, Germany), dried, and resolved in molecular biology-grade water. cDNA was obtained using the High Capacity Reverse Transcription Kit (ThermoFisher, Waltham, MA, USA). Gene expression was analyzed in Real-Time PCR with the use of Fast SYBR Green Master Mix (ThermoFisher). Primers were designed manually and the efficiency of primers was checked using the standard curve. For all sets of primers, the efficiency was close to R = 99%. The specificity of Real-Time PCR was checked with the use of melting curve analysis. The sequences and efficiency data of primers were published previously [21 (link),22 (link)]. Normalization was carried out to the housekeeping gene (β-actin) and calculated according to the ΔΔCt algorithm.