Ab 7900ht software

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.

Tissue and cellular RNA was extracted using TRIzol (Invitrogen), and total RNA (0.5 μg) was reverse-transcribed into cDNA according to the manufacturer’s instructions. For the quantitative real-time polymerase chain reaction (qRT-PCR) assays, the linearity of amplification of the test genes from cDNAs was established in preliminary experiments. The test genes included fatty acid synthesis genes (SREBP1c, LPL, C/EBPa, SCD1, HSL, FasN, mTOR, and PEPCK), glucose uptake genes (GLUT4, GLUT2, and IRS-1), macrophage marker (F4/80, CD11c and CD206), inflammatory cytokine genes (TNFα, IL1β, IL6, and MCP1), the anti-inflammatory cytokine gene IL10, and GAPDH (used as an internal control). cDNAs were amplified by real-time PCR in duplicate using a SYBR premix Ex Taq kit (Applied Biosystems, Foster City, CA, USA) and Thermal Cycler Dice (Life Technologies, Carlsbad, CA, USA). All reactions were performed in the same manner: 95°C for 10 seconds, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The results were analyzed using the real-time system AB 7900HT software (Life Technologies), and all values were normalized to the level of GAPDH.

Total RNA was extracted using an RNA Extraction 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) on a Bio-Rad CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA). The A/PR/8 Matrix (M) gene was detected using qRT-PCR. The following primers were used: M forward primer 5′- AATCCTGTCACCTCTGACTAAGG-3′, M reverse primer 5′-CATTYTGGACAAAKCGTCTACG-3′, and probe primer 5′-TGCAGTCCTCGCTCAC-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 60 °C for 45 s. The results were analyzed using the real-time system AB 7900HT software (Life Technologies, Carlsbad, CA, USA), and all values were normalized to GAPDH levels.