Cel mir 39 mimic

Circulating miRNAs were extracted from 200 μl of serum using an miRNeasy serum/plasma kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Briefly, serum samples were lysed with 1 ml of Qiazol and spiked with 3.5 μl of synthetic cel-miR-39 mimic (1.6 × 10⁸ copies/μl; Qiagen) to test extraction efficiency. Chloroform (200 μl) was used for phase separation at 4 °C and 10,000 rpm. The aqueous phase (600 μl) was added to 900 μl of 100% ethanol (Merck Millipore, MA, USA) and subsequently transferred into an RNeasy MiniElute spin column. These columns were washed with RWT buffer, RPE buffer, and 80% ethanol (Merck Millipore). All samples were eluted in 14 μl of RNase-free water.

Total RNA from cells or tissue was isolated using TRIzol Reagent and was reverse transcribed using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Grand Island, NY, USA). cDNA samples were amplified by using SYBR green PCR Master Mix (Thermo Fisher Scientific) with gene specific primers (Supplementary Information Table 2). 18S RNA was used as an internal control. RNAs from serum were isolated with mirVana PARIS Kit (Thermo Fisher Scientific) in conjunction with the synthetic spike-in control (cel-miR-39 mimic, QIAGEN, Hilden, Germany). Reverse transcription reactions for miRNA are performed using the TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific) and miRNA-specific stem-loop primers (Thermo Fisher Scientific). The RT products of serum were pre-amplified (Thermo Fisher Scientific) prior to the real-time PCR step to potentially enhance sensitivity. Real-time PCR reactions for miRNA are performed using the TaqMan 2X Universal PCR Master Mix II (Thermo Fisher Scientific) and TaqMan miRNA-specific probe mix (Thermo Fisher Scientific). Relative quantities of miRNAs were calculated by using the 2^-ΔΔCt method with U6 snRNA (Thermo Fisher Scientific) as the endogenous control. The assays were listed in Supplementary Information Table 3.