Mircury lna mirnas pcr assays

For the validation phase, the 13 deregulated miRNAs identified in the discovery phase were evaluated in serum samples from 23 healthy controls and 53 ALS patients. miRCURY LNA miRNAs PCR Assays (Qiagen) were used for each of the miRNAs, and amplified fragments were detected using ExiLENT SYBR Green master mix (Qiagen). In total, we studied 13 candidate miRNAs (miR-107, miR-142-3p, miR-30c-5p, miR-335-5p, miR-421, miR-423-3p, miR-454-3p, miR-7a-5p, miR-122-5p, miR-125a-5p, miR-30b-5p, miR-30e-5p, miR-2110), 2 haemolysis controls (miR-451a and miR-23a-3p), and 4 exogenous controls (spike-ins UniSp2, UniSp4, UniSp5 and UniSp6), as described above.

For each ddPCR assay, 9 μl cDNA sample (diluted 1:20), 10 μl 2X ddPCR supermix for Eva Green (Bio-Rad, Hercules, CA, United States), 1 μl of LNA PCR primers set was mixed (miRCURY LNA miRNAs PCR assays, Qiagen, Hilden, Germany). The mixture and 70 μl of droplet generation oil for Eva Green were respectively loaded into the sample and oil wells of a disposable droplet generator cartridge (Bio-Rad, Hercules, CA, United States). Therefore, droplets were generated by QX200 droplet generator device (Bio-Rad) and transferred to a 96 plate (Bio-Rad, Hercules, CA, United States). The cycling condition were 95°C for 5 min and 40 cycles (95°C for 30 s 58°C for 1 min) 4°C for 5 min and 90°C for 5 min. At the end of the PCR reaction, droplets were loaded in the QX200 droplet reader and analyzed using Quantasoft version 1.7.4 software (Bio-Rad, Hercules, CA, United States). A no template control (NTC) was included in every assay. miRCURY LNA miRNAs PCR assays used are: miRNA-26a, miRNA-26b, miRNA-30b, miRNA-30c (Qiagen, Hilden, Germany).

Total circulating RNA (tRNA) was isolated from patients’ serum using the miRNeasy Serum/Plasma Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Quality and quantity of microRNAs were assessed with NanoDrop™ 2000/2000c (Thermo Scientific, Waltham, MA, USA). Equal amounts (5 ng) of miRNA were used for reverse transcription (RT) using the miRCURY LNA RT Kit (QIAGEN, Hilden, Germany) and, for amplification by qPCR, using the miRCURY LNA SYBR^® Green PCR Kit (QIAGEN, Hilden, Germany) with CFX Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA). Briefly, hsa-let-7a-5p, hsa-let-7b-5p, hsa-miR-23a-3p, hsa-miR-27a-3p, hsa-miR-15a-5p, hsa-miR-320b, and hsa-miR-495-3p miRNAs were evaluated in duplicate for each sample and compared with hsa-miR-423-3p, identified as the most stably expressed and used as a reference. The miRNAs were selected from miRCURY LNA miRNAs PCR assays (QIAGEN, Hilden, Germany), and the miRBase accession numbers are reported in Table 1. The expression was calculated according to the 2^−ΔΔCt method.