Rotor gene q series software

Both the PCR and HRM steps were performed on the Rotor-gene Q (Qiagen, Duesseldorf, German). PCR was performed in 20 μL volumes containing 1.5 mmol/L Mg²⁺, 200 μM of each dNTP, 0.5 U of rTaq polymerase (Takara, Tokyo, Japan), 50 pmol Syto9 (Invitrogen, Waltham, US) and genomic DNA range was from 22 ng to 259 ng. The primer concentrations were optimized individually for the specific SNP amplicons. In the duplex PCR, the concentration of each rs662 primer was 0.15 μM, and 0.35 μM of each rs1045642 primer. In the triplex PCR, the concertation of each rs4244285 primer was 0.35 μM, 0.1 μM of each rs4986893 primer, and 0.35 μM of each rs12248560 primer. The PCR amplification protocol began with an initial denaturation at 95 °C for 3 minutes, followed by 35 cycles of 15 seconds denaturation at 95 °C and 30 seconds annealing-extension at either 62 °C (for the duplex PCR) or 60 °C (for triplex PCR). After amplification, the samples were cooled to 55 °C then melted from 55 °C to 95 °C at the melting rate of 0.2 °C/second. The melting curves were analyzed by Rotor-gene Q Series Software (Qiagen, Duesseldorf, German). Standard genotype reference samples of each genotype in each SNP sites were confirmed by Sanger Sequencing. The sample genotypes were determined by greatest similarity of their melting curves to those of standard genotype reference samples.

cDNA was synthesized from 2 µg total RNA using Maxima First Strand cDNA synthesis kit for RT-qPCR (Thermo Fisher Scientific), according to the manufacturer’s instructions. The gene expression levels were analyzed by quantitative reverse-transcription PCR using SYBR Green (TOPreal qPCR premix, Enzynomics) and a Rotor-Gene Q instrument (Qiagen) or Bio-Rad CFX Connect Real-Time System (Bio-Rad). The expression levels of gene transcripts were normalized to GAPDH (human) or 18S rRNA (mouse), and the results were evaluated by the Rotor-Gene Q series software (Qiagen) or Bio-rad CFX Maestro (Bio-Rad).

Total RNA was isolated using the DNA/RNA Shield (Zymo research) and the innuPREP RNA mini kit (Analytik Jena). cDNA was synthesized with innoScipt reverse transcriptase (Analytik Jena). Real time-PCR analysis of IL-15 and IL-18 was performed using innuMIX quantitative PCR (qPCR) MasterMix SyGreen (Analytik Jena). Oligonucleotide sequences were ordered at Biomers as follows: for β-actin, 5′-AAATCGTGCGTGACATCAAA-3′ and 5′-CAAGAAGGAAGGCTGGAAAA-3′; IL-15, 5′-CATTTTGGGCTGTGTCAGTG-3′ and 5′-TCTTCAAAGGCTTCATCTGCAA-3′. For the detection of mouse IL-18 Mm-Il18-1-SG QuantiTect primer assay was purchased from Qiagen. The quantitative mRNA levels were determined by using Rotor-Gene Q series software (Qiagen) and were normalized to the β-actin mRNA expression levels.