We kept the blood samples in coated vials of ethylenediaminetetraacetic acid (EDTA) to assess the expression of TNF-α, IL-6 receptor (IL-6r), and CRP genes. Peripheral blood mononuclear cells (PBMC) were separated during density gradient centrifugation (Ficoll-paque, Miltenyi Biotec GmbH, and Germany) by Ficoll-Histopaque solution gradient (Ficoll-paque, Miltenyi Biotec GmbH, and Germany). Total RNA from PBMC cells was extracted by Trizol Reagent kit (YTzol pure RNA, Iran) and 1 µg of the extracted RNA was used for complementary DNA (cDNA) synthesis by Prime Script-RT Reagent kits (Takara Bio Ink. Tokyo, Japan). We purchased special primers from Metabion (Metabion, Germany) whose sequences are shown in Table 1 . Data were normalized to the rate of 18S rRNA expressing as housekeeping gene and then calculated based on fold change formula. All samples were conducted in three versions.
Primers sequences for RT-PCR amplification
| Gene name and symbol | Sequence (5′ → 3′) |
|---|---|
| TNF-α | F: 5′-GTCAACCTCCTCTCTGCCAT-3′ |
| R: 5′CCAAAGTAGACCTGCCCAGA-3′ | |
| IL-6 | F: 5′-GGTACATCCTCGACGGCATCT-3′ |
| R: 5′-GT GCCTCTTTGCTGCTTTCAC-3′ | |
| CRP | F: 5′-TTTTCTCGTATGCCACCAAG-3′ |
| R: 5′-TTTCC AATGTCTCCCACCAG-3′ | |
| 18 s rRNA | F: 5′-ACCCGTTGAACCCCATTCGTG A-3′ |
| R: 5′-GCCTCACTAAACCATCCAATCGG-3′ |
F forward, R reverse