Quantification of Adipocyte miRNAs

Total microRNAs (miRNAs) were extracted and purified from frozen adipocytes by using miRNeasy Mini Kit (Qiagen, Germany). Reverse transcription of the extracted miRNAs was performed by the miScript Reverse Transcription Kit (Qiagen, Germany). cDNA was diluted 1:3 in RNase-free water and then qPCRs were performed in triplicate using the miScript SYBR-Green PCR kit (Qiagen, Germany) on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) [56 (link),57 (link)]. MiScript Primer Assays specific for hsa-miR-126-3p, hsa-miR-223-3p, hsa-miR-124-3p, hsa-miR-155-5p and hsa-miR-132-3p and hsa-RNU6 were obtained from Qiagen, and the probe sequences are proprietary. miRNAs expression was calculated using the Ct method and normalized to the expression of housekeeping RNU6. Mature miRNA sequences and miRBase Accession are reported in Table 2.