Real-time quantitative PCR analysis of human cell-free circulating miRNAs expression in early rheumatoid arthritis

The second metacarpal head (MCP2) of the 20 ERA patients and 6 sex- and age- matched healthy controls were scanned by HR-pQCT. Then the 20 ERA patients were devided into two sub-groups: bone erosion group and non-erosion group detected by HR-pQCT. Peripheral blood samples were collected using Ethylenediaminetetraacetic acid (EDTA) at the day of fasting status of ERA patients as well as HCs and were centrifuged at 3000×g for 10 minutes at room temperature. Then, the plasma samples were aliquoted and centrifuged at 3000×g for an additional 10 minutes at 4oC to remove any remaining cellular debris. The plasma was immediately stored at –80oC until analysis. miRNA were extracted from peripheral blood samples using EDTA in ten treatment-naïve ERA patients with maximal erosion volume at MCP2 detected by HR-pQCT, another ten treatment-naïve ERA patients without erosion and six age- and sex-matched healthy controls (HCs) as indicated in the summary. miRNA profiling was performed by TaqMan RT-qPCR (Life Technologies) according to the manufacturer’s instructions. This method allows the simultaneous analysis of 377 unique miRNAs (Human Pools A V2.1) using 384-well Cards.