Miscript kit

RNA was isolated using Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and reversely transcribed into cDNA. The mirPremier microRNA isolation kit (Sigma) was used to isolate miRNAs, which were reversely transcribed using the miScript kit (QIAGEN, Germantown, MD, USA). Quantitative PCR was applied to detect circTMCO3, TMCO3, miR-515-5p, ITGA8, TNF-α, iNOS, IL-10, Arg-1 using SYBR Green (Beyotime). GAPDH and U6 snRNA were used as normalization controls. The 2^−∆∆Ct method was used to calculate their relative expression. Primers were listed in Supplementary Table 1.

In order to quantify mature miRNA transcript levels as well as precursor miRNA levels, the miScript kit was used (Qiagen, Germany). Reverse transcription (RT) was performed using 300–400 ng of total RNA and “HiFlex” RT Buffer, which allows detection of both microRNA and messengerRNA. Temperature settings were chosen according to the suppliers recommendations (37 °C for 1 h, 95 °C for 5 min). cDNA was diluted 1:4 in nuclease-free water and qPCRs were run in quadruplicates using the miScript SYBR Green Kit (Qiagen, Germany) on the Rotorgene Q instrument (Qiagen, Germany): 95 °C → 15 min, 40 cycles of 94 °C → 15 s, 55 °C → 30 s, 70 °C → 30 s. SYBR Green fluorescence was measured at 70 °C and 80 °C. Commercial primer assays (Qiagen, Germany) were used for mature miRNA quantification. In-house designed primer assays were used for precursor-miRNA quantification (primer sequences are listed in Supporting Table 1).

Selected genes deemed statistically significant (as described below in the Statistical analysis section) by microarray analysis or nCounter system were further assessed by qPCR. Total RNA (400 ng mRNA and 200 ng miRNA) isolated from cells were reverse transcribed into complementary DNA (cDNA) using the RT2 First Strand Kit (Qiagen) or miScript Kit respectively. Gene profiling was performed according to the manufacturer’s instructions using custom RT2-profiler PCR arrays (Qiagen). Reactions were prepared in 96-well plates and performed using a spectrofluorometric thermal cycler (Biorad iCycler; Hercules, CA). The relative expression of each gene was determined by using the comparative threshold (Ct) method [27 (link)].

Total mRNA was obtained from HUVECs or lung endothelial cells using TRIzol reagent (Thermo-Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocols. After measuring the mRNA concentration, cDNA or miRNA was generated using cDNA synthesis master mix (CellSafe, Yongin, Republic of Korea) or the miScript kit (Qiagen, Hilden, Germany), respectively, according to the manufacturer’s instructions. The StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR green qPCR premix (Enzynomics, Daejeon, Republic of Korea) was used for the analysis. See Supplementary Table S1 for the primer sequences used for quantification. IDH2 and β-actin primers were purchased from Bioneer. β-Actin or RNU6 was used as an internal control, and relative expression was calculated using the 2^−Δ/ΔCt method.

Total RNA was isolated from cells using TRIzol (Invitrogen) following the manufacturer's protocol. Complementary DNA (cDNA) was synthesized by reverse transcription of RNA using M-MLV Reverse Transcriptase according to the manufacturer's protocol (Invitrogen). For mature miRNA analysis, cDNA was synthesized by reverse transcription of RNA using the miScript kit (Qiagen). qRT-PCR was performed using a Bio-Rad CFX96 Real-Time C1000 Touch Thermal Cycler and the following cycling conditions: (1) 95°C for 15 min, (2) 40 cycles of 94°C for 15 sec, 55°C for 30 sec, 70°C for 30 sec (collection step). Please see Supplementary Table 1 for relevant primer sequences. Expression was measured with SYBR Green/ROX qPCR master mix (Thermo Fisher Scientific). No template and no Reverse Transcriptase (–RT) controls were implemented to ensure samples were not contaminated. Melt curve analysis and electrophoresis of amplified products were performed as well. Quantitative Comparative CT (ΔΔCT) analysis was used to analyze gene expression changes relative to GAPDH or U6. qRT-PCR data represent the average of at least three independent experiments. Each sample was measured with n ≥ 2 technical replicates per target gene per independent experiment.

Study personnel from the Mexico site were trained in the isolation and quantitation of microRNAs at the US site. Both sites employed the same study protocol for all assays. RNA was extracted from 200 μL of plasma using the miRNeasy serum/plasma kit (Qiagen). Purified RNA was converted to cDNA using the miScript II RT Kit (Qiagen) in 20 μL reaction volumes using the miScript HiSpec buffer. Real-time quantitative polymerase chain reaction (qPCR) was used to assess relative expression of candidate microRNAs using the miScript kit (Qiagen). Experiments were carried out using a 384-well (US) or 96-well (Mexico) plate format on a Bio-Rad CFX real-time PCR machine using the manufacturer’s recommended cycling conditions. A standard curve was constructed for each microRNA target using a series of five serial dilutions. Both sites obtained at least three replicates measures per sample for each microRNA target. MicroRNA expression levels were normalized using cel-miR-39 and the global geometric mean signal of all reliably detected microRNAs [20 (link), 21 (link)], and relative expression levels were calculated using the ΔΔCt method [22 (link)].