Qubit microrna assay kit

Total RNA was extracted from 600 μl plasma using MagnaZol™ cfRNA Isolation Reagent (Bioo Scientific) and from 200 μl plasma using miRNeasy Serum/Plasma Kit (QIAGEN) following manufacturers’ instructions. To confirm the presence of miRNAs, samples were quantified using the Qubit™ microRNA Assay Kit (Thermo Fisher).

Fasting paired whole blood and serum samples were collected from the same participants during clinical visits. Serum was collected in Vacuette serum separating tubes (Greiner Bio-One GmbH, Austria). Samples were centrifuged at 3500 rpm for 10 min, aliquoted and stored at − 80 °C until further use.
Small RNAs were isolated from 300 μL of serum using the miRCURY RNA Isolation Kits (Exiqon, Vedbaek Denmark) and quantified with the Qubit microRNA Assay Kit (Thermo Fischer Scientific, USA). The quality of the total small non-coding RNAs extracted was assessed with the Agilent Small RNA kit (Agilent Technologies, USA), by using the 2100 Bioanalyzer Instrument (Agilent Technologies, USA). Small non-coding RNAs were hybridized on the Affimetrix array platform and analyzed as previously described^{11 (link)}.

RNA extraction was performed from 200 µL of EV solution using the miRNeasy kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. The RNA concentration was assessed using the Qubit™ microRNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA size distribution and yield were analyzed using the Agilent 2100 Bioanalyzer with the Small RNA analysis kit (Agilent Technologies, Santa Clara, CA, USA).

Cf-miRNAs were isolated from 300 µL thawed plasma using the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s procotol. Total cf-miRNAs were quantified using the Qubit microRNA Assay Kit and the Qubit Fluorometer 3.0 (Thermo Fisher Scientific, MA, USA).

Total RNA was extracted from the retinal tissues of various developmental stages using NucleoSpin miRNA kit (Macherey–Nagel, Düren, Germany, Cat: 740971.50) following manufacturer’s instructions. The miRNA concentration was assessed using Qubit microRNA Assay Kit (ThermoFisher Scientific, MA, USA, Cat: Q32880). The RNA integrity number (RIN), an algorithm for judging the integrity of RNA samples, were evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA, Cat: G2939BA) following the manufacturing instruction of the RNA 6000 Nano kit (Agilent Technologies, Santa Clara, California, USA, Cat: 5067-1511) and RIN > 7 was considered acceptable. MicroRNAs was also determined using the Agilent Small RNA Kit (Agilent Technologies, Santa Clara, California, USA, Cat: 5067-1548) to have deeper view in the 10–40-nucleotide size range.

MiRNA was extracted from extracellular vesicles abound in cell culture medium using EV-miR extraction kit (Topgen Biotech, Kaohsiung, Taiwan) following the manufacturer’s protocol and finally eluted in 20 μL low Tris-EDTA buffer (TE buffer) for each sample. The miRNA concentration was quantified by a Qubit microRNA assay kit using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MS, USA).