Exosomal miRNA Purification and Analysis

The miRNeasy Mini kit (Qiagen, Hilden, Germany) was used for the purification and extraction of miRNAs from exosomes isolated from cell culture conditioned supernatants or adipocytes. The retro-transcription of extracted miRNAs was performed by using the miScript Reverse Transcription kit (Qiagen). The cDNA obtained was diluted 1:3 in RNase-free water from adipocytes, while the exosome-cDNA was used without dilution. The qPCR experiments were performed with the miScript SYBR-Green PCR kit (Qiagen), as previously reported [38 (link)]. Signals were detected on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). MiScript Primer Assays specific for hsa-miR-34a-5p (MIMAT0000255), hsa-miR-155-5p (MIMAT0000646) and hsa-let-7c-5p (MIMAT0000064), hsa-SNORD6 and hsa-RNU6 were obtained from Qiagen. MiRNA expression was calculated using the C_T method and normalized to the expression of housekeeping genes SNORD6 for adipocyte-derived miRNAs, and Caenorhabditis elegans miR-39 (Cel-miR-39) for exosome-derived miRNAs (exo-miRNAs).