Higene total rna prep kit

The cells were stimulated with LPS (100 ng/mL) for 3 h in the presence or absence of peptide samples, and the total RNA was extracted using a HiGene™ Total RNA Prep Kit (BIOFACT, Daejeon, Korea). cDNA was synthesized from 500 ng of total RNA using a SuperiorScript III cDNA synthesis kit (Enzynomics, Daejeon, Korea). Real-time PCR was then performed on a Lightcycler 96 instrument (Roche Diagnostics Korea, Seoul, Korea), with a Dyne qPCR 2X PreMIX (Dyne Bio, Seongnam, Korea) and the following gene-specific primers (sense and antisense primer sequences, respectively): 5′-TGGACCTTCCAGGATGAGGACA-3′ and 5′-GTTCATCTCGGAGCCTGTAGTG-3′ for IL-1β, 5′-TACCACTTCACAAGTCGGAGGC-3′ and 5′-CTGCAAGTGCATCATCGTTGTTC-3′ for IL-6, and 5′-GGTGCCTATGTCTCAGCCTCTT-3′ and 5′-GCCATAGAACTGATGAGAGGGAG-3′ for TNF-α. The resulting 2^−ΔΔCt value for each group was used to calculate the relative expression ratio of the detected mRNA.

RNA was isolated using the HiGene Total RNA Prep Kit (Biofact, Daejeon, Korea) according to the manufacturer’s protocol. RNA concentration (A260) and purity (A260/A280 ratio) was measured by spectrophotometry (NanoDrop One Microvolume UV–Vis Spectrophotometer, Thermo Fisher Scientific, Dreieich, Germany), and the ratio for pure RNA A260/280 was higher than 2.0. The total RNA was reverse transcribed into cDNA using a cDNA kit (Qiagen, Hilden, Germany). CFX Connect Real-Time PCR Detection System (1855201, Bio-Rad Laboratories, Hercules, CA, USA) and CFX manager software (1845000 version 3.1, Bio-Rad Laboratories, Hercules, CA, USA) were used to quantify proinflammatory cytokine mRNA expression for RT-qPCR. qRT-PCR experiments were performed using specific forward and reverse primers (Table S1) and the conditions were as follows: 15 min at 95 °C, 20 s at 95 °C/40 s at 55 °C for 40 cycles, and 10 s at 95 °C/5 s at 65 °C/60 s at 95 °C for the melting curve. The qRT-PCR reaction system was 20 µL: SYBR Premix (Biofact)I, 10 µL; PCR Forward Primer (10 µM), 1 µL; PCR Reverse Primer (10 µM), 1 µL; cDNA template, 2 µL; and distilled water, 6 µL. The mRNA expressions of IL-6, IL-1β, and TNF-α were calculated by the 2^−ΔΔCT method with the internal reference as GAPDH.

To determine viral transcripts levels, total RNA was extracted using a HiGene™ Total RNA Prep Kit (Biofact, Daejeon, Republic of Korea). RNA was reverse-transcribed using a TOPscript™ cDNA synthesis kit (Enzynomics, Chungcheong-do, Republic of Korea) to synthesize complementary DNA (cDNA), which was amplified using the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with HOT FIREPol^® EvaGreen qPCR mix Plus (Solis BioDyne) and specific primers. The primer sequences used for amplification were as follows: IAV M1, 5′-GACCAATCCTGTCACCTCTGAC-3′ (forward) and 5′-AGGGCATTTTGGACAAACCGTCTA-3′ (reverse); Canis lupus familiaris glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CCAGGGCTGCTTTTAACTCTGG-3′ (forward) and 5′-ACTGTGCCGTGGAATTTGCCG-3′ (reverse).