4.6. Real-Time PCR Analysis

Total RNA was isolated by using TRI^® Reagent (Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacturer’s instructions. The synthesis of single-strand cDNA was carried out on 1 μg of RNA, using iScript™ Reverse Transcription Supermix (BIO-RAD, Milan, Italy) following manufacturer’s instructions. Real-time PCR was performed with the StepOnePlus^TM Real-Time PCR System (Thermo Fisher, Carlsbad, CA, USA) using iQ SYBR Green Supermix (BIO-RAD, Milan, Italy). A non-template control (NTC) was added in order to exclude the presence of genomic DNA, and all experiments were performed in triplicates. Samples were analyzed with the 2^ΔΔCt method. The mRNA levels of target genes were normalized with 18S ribosomal RNA for 3T3-L1 cells, and GAPDH for hADSCs. The primers were designed using Oligo Perfect^® Designer Software (Invitrogen). The nucleotide sequences of the primers utilized are reported in Table 1 and Table 2.

Primer sequences for Mouse 3T3L1 cells.

Primer sequences for human Adipose Derived Mesenchymal Cells.