Reverse transcription reagent

Cells isolated from whole constructs were used for RNA isolation using ReliaPrep RNACell Miniprep System (Promega, Madison, WI) according to the manufacturer's protocol. RNA (1000 ng; or 500 ng for low-yield RNA samples) was reverse transcribed to cDNA using reverse transcription reagents (Go Script, Promega, Madison, WI) as per the manufacturer's protocol. A total volume of 0.4 µl of cDNA (0.8 µl of cDNA for low-yield RNA/cDNA samples) was used per 20 µl real-time qPCR reaction using Power Up SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and run for 40 cycles in triplicates on a 7500 Fast Real Time PCR System machine (Applied Biosystems), according to the manufacturer's instructions. Gene-specific primers are listed in Table S7. Relative gene expression, normalized to β-actin (ΔCT) and control VF mucosae (ΔΔCT), was calculated as fold change using the 2^−ΔΔCT method. If undetected, a cycle number of 40 was assigned to allow fold change calculations. Data are presented as the average of the three biological and technical replicates (for 5% ECVE) and three biological and technical replicates (for 0.5% ECVE) ±s.e.m. of the mean. One-way ANOVA with Tukey’s HSD test were used to confirm statistical significance in gene expression, *P≤0.05.

Total RNA was isolated from cells using a RNA kit (Qiagen). The RNA was reverse-transcribed into cDNA with reverse transcription reagents (Promega). Here, quantitative PCR (qPCR) and qPCR with reverse transcription systems (A6002, Promega) were used according to the manufacturer’s instructions. Gapdh was used as a reference gene to normalize other genes. A list of the primer sequences used for qPCR in this study is provided in Supplementary Table 5.

HCV RNAs were purified using Trizol LS Reagent (Ambion, Austin, TX, USA), according to the manufacturer’s protocol. Complementary DNA synthesis was performed using a reverse transcription reagent (Promega, Madison, WI, USA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using the TaKaRa SYBR Premix EX Taq II protocol with an IQ5 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The sequences of primers for qRT-PCR were as follows: 5′-TCTGCGGAACCGGTGAGTA-3′ and 5′-TCAGGCAGTACCACAAGGC-3′.

BV2 cells were seeded in a 6-well plate at a density of 2.0 × 10⁵ cells/mL and treated with LOS-ME 25, 100, 200 μg/mL, and LPS 200 ng/mL. The total RNA from the treated cells was acquired using Trizol reagent (Invitrogen Life Technologies, Waltham, MA, USA) according to the manufacturer’s instructions. The cDNA synthesis was performed by measuring the concentration of total RNA using a NanoDrop machine (Molecular Devices, San Jose, CA, USA), followed by reverse transcription using reverse transcription reagent (Promega, Madison, WI, USA) and PCR equipment (Bio-Rad, San Francisco, CA, USA) according to the manufacturer’s instructions. Then, using specific primers, cDNA was amplified by PCR following the method described previously [2 (link)]. Electrophoresis was performed with a 1–2% agarose gel using Mupid-2plus (TaKaRa, Kyoto, Japan) bands were photographed with a Gel Imaging System (Davinch-K, Seoul, Korea) and then calibrated through ImageJ software.