Studies were approved by the Institutional Review Board of the University of Illinois at Chicago, and all donors gave informed written consent. RA patients were diagnosed according to the American College of Rheumatology 1987 revised criteria (23 (link)).
RNA was extracted from 1 ml of RA SF, osteoarthritis (OA) SF, RA plasma, and normal plasma. The RNA (10 ng) was reverse transcribed, and levels of miRNA were determined by real-time reverse transcription–polymerase chain reaction (RT-PCR) using a microRNA assay for hsa-let-7b according to the manufacturer’s instructions (Life Technologies). The copy number of exosomal miR-let-7b was calculated via a standard curve using the synthetic miR-let-7b (Gen-Script) and plotting the Ct values against the copy number, as described previously (24 (link)).
Mononuclear cells were isolated by Histopaque gradient centrifugation. Monocytes and T cells were isolated from normal and RA peripheral blood (PB) and/or SF using a negative selection kit (StemCell Technologies) (25 (link),26 (link)), and neutrophils were isolated from normal PB (27 (link)). The copy number of miR-let-7b and miR-29a (Life Technologies) in exosomes was determined in PB T cells, in vitro differentiated macrophages, and neutrophils, as well as in RA SF macrophages and RA ST fibroblasts.
RNA was extracted from 1 ml of RA SF, osteoarthritis (OA) SF, RA plasma, and normal plasma. The RNA (10 ng) was reverse transcribed, and levels of miRNA were determined by real-time reverse transcription–polymerase chain reaction (RT-PCR) using a microRNA assay for hsa-let-7b according to the manufacturer’s instructions (Life Technologies). The copy number of exosomal miR-let-7b was calculated via a standard curve using the synthetic miR-let-7b (Gen-Script) and plotting the Ct values against the copy number, as described previously (24 (link)).
Mononuclear cells were isolated by Histopaque gradient centrifugation. Monocytes and T cells were isolated from normal and RA peripheral blood (PB) and/or SF using a negative selection kit (StemCell Technologies) (25 (link),26 (link)), and neutrophils were isolated from normal PB (27 (link)). The copy number of miR-let-7b and miR-29a (Life Technologies) in exosomes was determined in PB T cells, in vitro differentiated macrophages, and neutrophils, as well as in RA SF macrophages and RA ST fibroblasts.
