Cel mir 39 1.6 108 copies spike in control

Total RNA was extracted from serum using miRNeasy serum/plasma kits (Qiagen, United States). A cel-miR-39 (1.6 × 10⁸ copies) spike-in control (Qiagen, United States) was added to provide an internal reference. cDNA conversion was performed with 10 ng of total RNA using TaqMan microRNA reverse transcription kits (Applied Biosystems, United States). Amplification of miR-33a, miR-126, miR-499, miR-186 and miR-146a, was performed by quantitative real-time PCR using the TaqMan assay (Applied Biosystems, United States) and expression as normalized against cell-miR-39. The sequences and codes of the assessed miRNAs are listed in Supplementary Table 1 (Private sharing link for Figshare data https://figshare.com/s/2d858620da6b13fe2fec). Values were calculated by the 2^−(ΔΔCt) method.

To analyze the circulating microRNAs from serum, total RNA was extracted using the miRNeasy serum/plasma kit (Qiagen, USA). Then, cel-miR-39 (1.6×10⁸ copies) spike in control (Qiagen, USA) was added to provide an internal reference. cDNA conversion was performed from 10ng of total RNA using the TaqMan microRNA Reverse Transcription kit (Applied Biosystems, USA). Analysis of the gene expression of miR-122, miR-145 and miR-143, together with the cell-miR-39 normalizer, was performed by RT-qPCR using TaqMan assay (Applied Biosystems, USA). The sequences and codes of the assessed microRNAs are described in Supplementary Table 1. Values were calculated by formula 2 ^−(ΔΔCt).