Rnase free dnaase 1

Total RNA was extracted using Trizol reagent, and the RNA obtained was treated with RNAse-free DNAase I (Invitrogen Life Technologies) according to the manufacturer's protocol. The complementary DNA was synthesized from 1 μg of total RNA using superscript first-strand synthesis system (Invitrogen Life Technologies). Real-time PCR was performed using SYBR-green fluorescence quantification system in a Step-One real-time PCR 96-well plate (Applied Biosystems, Warrington, UK). Each cDNA sample (2.5 μl) was used as a template for the PCR amplification mixture containing forward and reverse primers (900 mM each). Reaction mixtures were subjected to the following amplification scheme: one cycle at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 56°C for 1 min. Relative gene expression was calculated using the ^2-ΔΔCt method, and the expression data were normalized with endogenous β-actin. The sequences of primers used for IFN-γ were 5′ -GGCATTTTGAAGAATTGGAAAG-3′ (forward) and 5′ -TTTGGATGCTCTGGTCATCTT-3′ (reverse) as previously described16 (link).The primers used for β-actin were 5′-TGCCGACAGGATGCAGAAG-3′ (forward) and 5′ -CTCAGGAGGAGCAATGATCTTGA-3′ (reverse) as previously described17 . All real-time PCRs were performed in triplicate.

The total RNA was extracted from the peripheral blood mononuclear cells (PBMCs) of T2DM patients using Trizol, and the RNA obtained was treated with RNase-free DNAase I (Invitrogen Life Technologies, Carlsbad, CA). Genomic DNA was isolated from blood cells using the DNeasy Blood and Tissue Kit (Qiagen, Germany), according to the manufacturer's instructions. The nucleic acid samples were quantified by measuring the absorption at 260 nm.