Cfx96 thermal cycler

Total RNA was extracted from Vero cells and pancreata of mice using a QIAamp^® viral RNA mini kit (Qiagen, Hilden, Germany). Taqman real-time PCR and reverse transcription PCR were carried out using AgPath-ID™ One-Step RT-PCR Reagents (Applied Biosystems, Waltham, MA, USA) and a Bio-Rad CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA). The EV71 5′ noncoding region (NCR) of the gene was detected using qRT-PCR. The following EV71 5′NCR primers were used: forward primer 5′-GCGATTGTCACCATWAGCAGYCA-3,’ reverse primer 5′-GGCCCCTGAATGCGGCTAATCC-3,’ and probe primer 5′-CCGACTACTTTGGGWGTCCGTGT-3′. The following GAPDH primers were used: forward primer 5′-GGTCTCCTCTGACTTCAACA-3′, reverse primer 5′-AGCCAAATTCGTTGTCATAC-3′, and probe primer 5′-CCCTCAACGACCACTTTGTCAAG-3′. The cycling conditions were as follows: heating at 45 °C for 10 min for reverse transcription, reverse transcription inactivation, and initial denaturation at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s and at 62 °C for 45 s. The results were analyzed using the real-time system AB 7900HT software (Life Technologies) and all values were normalized to GAPDH levels. A Bio-Rad CFX96 thermal cycler was used at the Core Facility for Innovative Cancer Drug Discovery (CFICDD) at Kangwon National University.

After incubation, cells were centrifuged at 300× g for 10 min after which the supernatant fluid was carefully removed. RNA was isolated from each cell pellet using AllPrep DNA/RNA/miRNA Universal Kit (Quiagen, Hilden, Germany). RNA concentration was measured using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using the miScript II RT Kit (Quiagen, Hilden, Germany), and conversion to cDNA was performed in the DNA Engine^® Thermal Cycler (BioRad, Hercules, CA, USA). cDNA was diluted in 40 µL RNase-free water. The PCR reagent consisted of 12.5 µL 2xQuantiTect SYBR Green PCR Master Mix, 7.5 µL RNase-free water, 2.5 µL 10x Primer Assay, and 2.5 µL Template cDNA. PCR was performed in a CFX96TM Thermal Cycler (BioRad, Hercules, CA, USA). Genes of interest were selected according to the following criteria: (i) genes that are major players and representative of the pathways described in Secs 1.1 and 1.2); (ii) genes that are known to be involved in two or more of these relevant suggested pathways; (iii) genes that are expected to undergo adaptive responses; (iv) genes coding a considerable number of proteins known to be regulated at the mRNA level.

The amplification reactions were carried out in a total volume of 10 μL, which consisted of 2 μL of DNA samples and 8 μL of the master mixture. The latter contained 500 nM of each primer, 5 μL of 2 × of the commercial q-PCR master mix SsoFast™ EvaGreen® Supermix (Bio-Rad, Hercules, CA, USA). Amplifications were performed on a CFX96^TM thermal cycler (Bio-Rad), comprising 1 cycle of 2 min at 98 °C (hot start) followed by 40 cycles of 98 °C for 5 s for denaturation. Annealing step was performed for 5 s at 58 °C and extension was made at 65 °C to 95 °C for 5 s (with temperature increments of 1 °C). The q-PCR products were analyzed by melting curves for unspecific products or primer dimer formation. Fluorescence was measured after each cycle. Each assay was carried out in duplicate and the average C_t value from each duplicate was used for analysis.