Nucleospin mirna plasma serum

The volume of 300 µL of sera from PsAs, AS, RA and HC was used to isolate circulating miRs using NucleoSpin^® miRNA plasma/serum (Macherey–Nagel, Düren, Germany), according to the manufacturer’s protocol. miR was extracted with 40 uL of ultra-pour water. TaqMan^® microRNA RT Kit was used to reverse transcribe to cDNA with the use of TaqMan^® MicroRNA Assays for hsa-miR-222-3p (471527_mat), hsa-let-7d-5p (000377), hsa-miR-223-5p, hsa-miR-146b-3p, hsa-miR-10b-5p, hsa-miR-26a-2-3p, hsa-miR-92b-3p, and hsa-miR-485-3p. Based on previously published data, miR-16-5p (477860_mir) was used to normalise circulating miR expression [11 (link)]. TaqMan^® miRNA Assays and TaqMan^® Universal PCR Master Mix II no UNG were used to quantify miR expression by quantitative polymerase chain reaction (qPCR) using QuantStudio 5 qRT-PCR machines (all from Thermo Fisher Scientific, Waltham, MA, USA). The expression levels of miRs relative to the average HC (arbitrarily set at 1) were calculated using the following equation: 2^−ΔΔCT.

The volume of 300 µl of sera from HC, RA and AS patients was used to isolate circulating miRNA-5196 using NucleoSpin® miRNA plasma/serum (Macherey–Nagel, Germany) according to the manufacturer’s protocol. TaqMan ® microRNA RT Kit (Thermo Fisher Scientific, USA) was used to reverse transcribe to cDNA with the use of TaqMan® MicroRNA Assays for hsa-miR-5196-5q (471527_mat), and hsa-let-7a (000377) all from Thermo Fisher Scientific (USA). Based on previously published papers, hsa-let-7a was used as an internal control to normalize miRNA-5196 input (Davoren et al. 2008 (link); Li et al. 2015 ). The 20 µL PCR reaction included 1.33 µL RT product, 1× TaqMan Universal PCR master mix and 1 µL primers and probe mix of the TaqMan MicroRNA Assay Kit (Thermo Fisher Scientific, USA). Reactions were performed at 95 °C for 10 min, followed by 50 cycles at 95 °C for 15 s and 60 °C for 1 min. Samples were analysed in triplicate using the Viia 7 or QuantStudio 5 qRT-PCR machines (Thermo Fisher Scientific, USA). The expression levels relative to the average HC (arbitrarily set at 1) were calculated using the following equation: (2^Delta Delta CT) − 1, all normalized to hsa-let-7a internal control.

The volume of 300 µl of sera or plasma from SSc (n=10), RA (n=44) patients and and HC (n=37) patients was used to isolate circulating miRNA using NucleoSpin^® miRNA plasma/serum (Macherey–Nagel, Germany) according to the manufacturer’s protocol. Circulating miRNA expression level was validated using TaqMan ^® microRNA RT Kit (Thermo Fisher Scientific, USA) and TaqMan^® MicroRNA Assays. The IDs of the probes are: hsa-miR-589-3q (479072_mir), hsa-3614-5p (478836_mir), hsa-miR-374a-5p (478238_mir), hsa-miR-29c-3p (479229_mir), hsa-miR-19b-3p (478264_mir), hsa-miR-503-5p (478143_mir), has-miR-16-5p (477860_mir), all from ThermoFisher Scientific (USA). The expression levels of circulating miRNA in SSc and RA patients were relative to the average HC (arbitrarily set at 1) and were calculated using the following equation: 2^(-delta delta CT). All samples were normalized to miRNA-16-5p as an internal control.