Rotor gene q 5plex high resolution melting

Total RNAs and miRNAs were extracted from sera samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, and RNA concentration and purity were evaluated spectrophotometrically at 260 and 280 nm. RNA integrity was visually confirmed by agarose gel electrophoresis. Synthesis of cDNA was completed by reverse transcription reaction using a miScript RT Kit (Qiagen, Hilden, Germany). In the second step, cDNA was amplified for miRNA expression using a miScript Primer Assay for miRNA amplification (Rn_miR-137_1 and Rn_miR-106b-5p_1 miScript Primer Assay). The At_U19_1 and Rn_ GAPDH genes were used as reference housekeeper genes. The thermal cycling was adjusted according to the manufacturer’s instructions. The PCR analysis was conducted on a Rotor-Gene Q 5plex high resolution melting (HRM) platform (Qiagen, Hilden, Germany). The fluorescence data were collected at the extension step. Following amplification, gene expression was calculated using the 2^∆∆Ct method.

A total of 131 EDTA containing blood samples were collected from patients with asthma and stored at −80°C until nucleic acid purification. Residual EDTA containing blood samples collected for routine biochemistry tests were used as the control group. Nucleic acids were isolated from these samples using a commercial genomic DNA extraction kit (Thermo Scientific, MA, USA).
The Rotor-Gene Q 5plex high resolution melting (HRM) platform and Rotor-Gene 6000 series software v1.7 (Qiagen, Hilden, Germany) were used for real time PCR amplification and HRM analysis. 5x Hot Fire Pol Evagreen HRM mix (Solis BioDyne, Tartu, Estonia) used for PCR amplification and PCR reactions were performed in a 20 μL final volume containing 5 μL of purified genomic DNA. 0.1 mM of primers (Table 1) used for amplification. Amplification was performed 95°C for 10 minutes for initial denaturation followed by 30 cycles at 95°C for 10 seconds, 53°C for 45 seconds and 72°C for 45 seconds. After amplification, denaturation was performed from 72°C to 90°C for HRM analysis. Representative samples of genotypes were confirmed by DNA sequencing for TLR4 polymorphisms and via the restriction fragment length polymorphism (RFLP) method for the CD14 polymorphism. Representative samples were also used controls for further tests (Table 1) [14 (link),17 (link)].