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Serum extracelluar vesicles miRNA in premature neonates with chronic lung disease (CLD) and non CLD

ABSTRACT: Serum were obtained from 4 groups, day0 and day28 neonates with CLD and non-CLD. Serum samples (250ul) from cord blood at birth and venous blood of neonates at 28 DAYS were transferred to sterile tubes containing 63 μl of ExoQuick precipitation solution (System Bioscience, Mountain View, CA, USA), and mixed. The mixtures were incubated for 30 min at 4℃, and centrifuged at 1500×g for 30 min. Then, the supernatants were removed, the pellets (extracellular vesicles) were diluted to 60 μl with phosphosaline buffer, and then thawed on dry ice for total RNA extraction with a miRvana isolation kit (Ambion). 3pM synthetic ath-miR159a was spiked into each sample to correct for technical differences. Using a human miRNA array, we identified 62 miRNAs that were universally expressed in CLD patients and non-CLD patients (Normalized Template).

ORGANISM(S): Homo sapiens

PROVIDER: GSE129400 | GEO | 2021/04/06

REPOSITORIES: GEO

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Project description:Analysis of polar metabolites Serum samples were randomized and sample preparation was carried out as described previously (Castilloet al. 2011). In summary, 400 μL of MeOH containing ISTDs (heptadecanoic acid, deuterium-labeled DL-valine, deuterium-labeled succinic acid, and deuterium-labeled glutamic acid, c = 1 µg/mL) was added to 30 µl of the serum samples which were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) and 350 μL of the supernatant was collected after centrifugation. The solvent was evaporated to dryness and 25 μL of MOX reagent was added and the sample was incubated for 60 min at 45 °C. 25 μL of MSTFA was added and after 60 min incubation at 45 °C 25 μL of the retention index standard mixture (n-alkanes, c=10 µg/mL) was added. The analyses were carried out on an Agilent 7890B GC coupled to 7200 QTOF MS. Injection volume was 1 µL with 100:1 cold solvent split on PTV at 70 °C, heating to 300 °C at 120 °C/minute. Column: Zebron ZB-SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 µm. With initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C at 20 °C/min and then to 300 °C at 40 °C/min (5 min). EI source: 250 °C, 70 eV electron energy, 35µA emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, methionine, ornithine, phenylalanine, serine and threonine purchased from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 μg/mL. An aliquot of each sample was collected and pooled and used as quality control samples, together with a NIST SRM 1950 serum sample and an in-house pooled serum sample. Relative standard deviations (% RSDs) of the metabolite concentrations in control serum samples showed % RSDs within accepted analytical limits at averages of 27.2% and 29.2% for in-house QC abd pooled QC samples.

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Serum extracelluar vesicles miRNA in premature neonates with chronic lung disease (CLD) and non CLD

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