Qrt pcr taqman microrna assays

Total RNA was purified from the isolated exosomes using miRNeasy kit (Qiagen) according to the manufacturer's instructions. Exosomal RNA was quantified and characterized by NanoDrop® Spectrophotometer (Thermo Scientific) and Bioanalyzer RNA nano chip (Agilent), respectively. Expression levels of miRNAs in the exosomes were validated using individual stem-loop quantitative RT-PCR (qRT-PCR) TaqMan microRNA Assays (Applied Biosystems) according to the manufacturer's instructions. We performed qPCR assays for 8 differentially expressed miRNAs (hsa-miR-4497, hsa-miR-133a, hsa-miR-1246, hsa-miR-1290, hsa-miR-518e-3p, hsa-miR-629–3p, and hsa-miR-200c-5p) for which advanced miRNA assays were available. We used a subset of both T1D and control samples (n = 4). hsa-let-7a-5p was used as control and all samples were tested in duplicates (Supplementary File 1, Figure S1). The expression levels of these miRNAs were also tested in the plasma of same mothers.

miR-133a was quantified using qRT-PCR TaqMan^® MicroRNA Assays (Applied Biosystems) in accordance with the manufacturer’s instructions, and normalized with U6 small nuclear RNA (snRNA) calculated by means of the 2^−ΔΔCt method53 (link) (n = 6).