Seramir exosome rna amplification kit

To detect the exosomal miRNAs, exosomes were eluted from magnetic beads by incubation in elution buffer for 2 h on a rotating rack. Total RNA from the exosomes was enriched using the SeraMir ExosomeRNA Amplification kit (System Bioscience). The purified RNA yield was determined by measuring the absorbance at 260 nm using an SSP-3000 Nanodrop spectrophotometer (Infinigen Biotech, City of Industry, CA, USA), and RNA quality was evaluated with an Agilent Bioanalyzer (2100; Agilent Technologies, Palo Alto, CA, USA).

Serum was collected from the included subjects. It was aliquoted in 1 mL tubes and stored at −80°C for subsequent exosome isolation. Serum exosome was extracted using ExoQuick (System Biosciences). Exosomal RNA was isolated from the serum using SeraMir™ Exosome RNA Amplification Kit (System Biosciences).

RNA from serum‐derived EVs was extracted using the SeraMir™ Exosome RNA Amplification Kit (System Biosciences) according to the manufacturer's instructions. Briefly, serum samples (300 μL) were mixed with ExoQuick solution (72 μL) and incubated at 4 °C overnight. The mixtures were centrifuged at 1500 g for 30 min at room temperature before the supernatants were removed and the pellets resuspended in PBS (100 μL). The EV lysates were mixed with lysis buffer (300 μL) and 100% EtOH (200 μL). After vortexing for 10 s, the mixtures were transferred to a spin column and centrifuged at 15 928 g for 1 min and then washed twice with wash buffer (400 μL). After further centrifugation for 2 min, small EV‐derived RNA was eluted in elution buffer (30 μL).
Serum RNA was isolated using the TRIzol‐LS reagent (Invitrogen). In brief, serum (300 μL) was lysed in TRIzol‐LS (900 μL) before the RNA was phase separated using chloroform (240 μL), precipitated with 100% isopropanol, and washed in 75% EtOH. Finally, the RNA was eluted in RNase‐free water (30 μL).
The RNA concentration was assessed using the NanoDrop 2000 spectrophotometer (Thermo Scientific), while its yield and size distribution were analyzed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano kit (Agilent Technologies, Foster City, CA, USA).

Total RNAs were extracted from cultured EECs or isolated EVs using the ISOGEN reagent (Nippon Gene, Tokyo, Japan) or SeraMir Exosome RNA Amplification Kit (System Biosciences), respectively, according to the manufacturer’s protocols. Reverse-transcription of the isolated RNAs was performed with ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), which was then subjected to qPCR amplification using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific). For miRNAs analysis, the miRNA First-Strand Synthesis Kit (Clontech, Tokyo, Japan) was used to synthesize micro-cDNA, then subjected to qPCR by using the SYBR Green (Clontech). Primers are listed in Table S1. Calibration curves were used to examine amplification efficiencies of each target gene and the reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), and U6, and were found to be comparable. Sequence Detection System software v2.3 (Applied Biosystems) was used to determine average threshold (Ct) values for each target^{39 (link),40 (link)}.

Adipocyte-derived EVs were isolated using the commercially available EoxQuick Precipitation Solution (System Biosciences, Mountain View, CA) from the serum of an all-female subset, chosen to be phenotypically representative of the larger cohort, as previously described [15 (link)]. Total RNA was extracted from adipocyte-derived EVs using the commercially available SeraMir Exosome RNA Amplification Kit (System Biosciences, Mountain View, CA) according to manufacturer instructions. RNA was labeled with Affymetrix^® FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA) according to standard procedures. Labeled RNA was hybridized to Affymetrix GeneChip microRNA 4.0 arrays and run using a Fluidics Station 450 Protocol (FS450_002) (Affymetrix, Santa Clara, CA). MicroRNAs and ProbeIDs used for statistical analysis are provided in Additional file 2: Table S1 (Accession #: GSE125494).