RT-PCR experiments to measure IL-6 and OXTR mRNA expression

RT-PCR was used to quantify cellular OXTR, and cytokine mRNA expression levels. Total RNA was isolated using RNeasy extraction kit (Qiagen, Valencia, CA, USA), then cDNA was synthesized after DNase I treatment using reagents from Applied Biosystems (Foster City, CA, USA) following manufacturer’s instructions.

Quantitative gene expression of mouse OXTR, IL-6, adiponectin, macrophage marker, F4/80, leptin, and TNF-α by RT-PCR was performed with the TaqMan gene expression assay. The following Applied Biosystems inventoried primers were used: mouse Oxtr (Mm01182684_m1), mouse Il-6 (Mm00446190_m1), mouse adiponectin (Adipoq; Mm00456425_m1), F4/80 (Mm00802529_m1), and Tnf-α (Mm00443260_g1). cDNA (50 ng) were amplified with TaqMan Universal PCR Master Mix and reactions were run using universal cycling conditions on an Applied Biosystems 7500 Real-Time PCR system. Samples were analyzed in triplicate. The ΔΔCT (threshold cycle) method was used to analyze changes in gene expression. Relative quantification (RQ) was expressed as the fold change compared to the appropriate control condition [29]. 18S rRNA was used as the endogenous RNA control. A non-template control was performed to ensure that there was no amplification of genomic DNA.