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