4.2.12. Analysis of Inflammatory Gene Expression

We performed a quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate the expression of inflammatory genes, such as TNF-α, F4/80, CCL2, CCL4, CCL5, and CXCR4. Prior to qRT-PCR, complementary DNA (cDNA) was synthesized using an Advantage RT PCR Kit (Clontech, Palo Alto, CA, USA). We extracted 1 μg of RNA from the epididymal fat pad, oligo (dT) and RNase-free H₂O were mixed, and the mixture was heated at 70 °C for 2 min. Next, we added a 5× reaction buffer, MMLV reverse transcriptase, recombinant RNase inhibitor, and 10 nM dNTP. Further, the mixture was incubated at 42 °C for 60 min and at 94 °C for 5 min. cDNA was obtained through reverse-transcription PCR: dH₂O, 2× SYBR reaction buffer, and primers were added, and qRT-PCR was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following primers were used: TNF-α, 5′-TTCTG TCTAC TGAAC TTCGG GGTGA TCGGT CC-3′, and 5′-GTATG AGATA GCAAA TCGGC TGACG GTGTGGG-3′; F4/80, 5′-CTTTGGCTATGGGCTTCCAGTC-3′, and 5′-GCAAGGAGGACAGAGTTTATCGTG-3′; CCL2, 5′-AGGTCCCTGTCATGCTTCTGG-3′, and 5′-CTGCTGCTGGTGATCCTCTTG-3′; CCL4, 5′-CTCAGCCCTGATGCTTCTCAC-3′, and 5′-AGAGGGGCAGGAAATCTGAAC-3′; CCL5, 5′-TGCCCACGTCAAGGAGTATTTC-3′, and 5′-AACCCACTTCTTCTCTGGGTTG-3′; CXCR4, 5′-TCAGTGGCTGACCTCCTCTT-3′, and 5′-CTTGGCCTTTGACTGTTGGT-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene), 5′-AGTCCATGCCATCACTGCCACC-3′, and 5′-CCAGTGAGCTTCCCGTTCAGC-3′. To analyze gene expression, we converted the threshold cycle (Ct) of each gene obtained by SDS Software 2.4 (Applied Biosystems^®, USA) to relative quantitation (RQ) with respect to values for GAPDH and calculated the fold change. The fold change value of the experimental group was adjusted with respect to that for the NC group that was considered to be 1.