Revertra ace α kit

After treatment with 40% CM or 0.125, 0.25, 0.5 and 1 μg/ml CD147, total RNA was extracted from the LX-2 cells using TRIzol reagent, according to the manufacturer’s instructions (Promega Corporation, Madison, WI, USA). cDNA was reverse transcribed from 1 μg total RNA, using ReverTra Ace-α™ kit (Toyobo Co., Ltd., Osaka, Japan). RT-qPCR analysis was performed using SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions, using a StepOnePlus™ Real-Time PCR system (Applied Biosystems). A total of 1.6 μl template cDNA was used for amplification, and the PCR conditions were set at: Initial denaturation at 95°C for 30 sec, 95°C for 5 sec and 58°C for 30 sec for 35 cycles, then 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and annealing and extension at 58°C for 30 sec. The gene expression levels of α-SMA, collagen I and TIMP were measured and compared against the expression of β-actin. The sequences of the oligonucleotides used are shown in Table I. The data were analyzed usign StepOne v2.3 software (Life Technologies, Carlsbad, CA, USA).

BV2 microglia cells were plated overnight in 6-well culture plates and were pretreated for 1 h with the indicated concentrations of SLCN (18 and 36 µg/mL) and base curcumin (36 µg/mL) before incubation in a medium containing LPS (100 ng/mL). The total RNA was extracted using TRIZOL (Invitrogen). RNA (1 µg) was reverse-transcribed using ReverTra Ace-α kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The inducible nitric oxide synthase, cyclooxygenase type 2 (COX-2), tumour necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β), IL-6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were amplified from the cDNA via polymerase chain reaction (PCR). cDNA was amplified by PCR using the specific primers mentioned in Table 2. PCR was performed using an initial step of denaturation (5 min at 94 °C), 20–27 cycles of amplification (94 °C for 30 s, 54–58 °C for 1 min and 72 °C for 1 min) and an extension (72 °C for 5 min). PCR products were analysed on 1.5% agarose gels with EtBr stained. The mRNA of GAPDH served as an internal control for sample loading and mRNA integrity. The band intensity was quantified via a densitometry analysis using multi-gauge software V3.1 (Fujifilm, Tokyo, Japan).

The tissue culture samples for RNA extraction were retrieved from −80°C storage and thawed for further processing. The viral RNA was extracted according to the given protocol (25 (link)). The extracted RNA was introduced for reverse transcription using a ReverTra Ace-α kit (Toyobo, Japan) in accordance with manufacturer’s directions. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was accomplished by SYBR Green Real-Time PCR Master Mix (Takara, Japan) in accordance with manufacturer’s directions. The mRNA expression levels were normalized relative to β-actin. Data were calculated as the fold difference in the treatment compared with the control groups. Viral cDNA was detected using the JEV C gene specific forward and reverse primers.

All of the 64 EV-A71 clinical specimens isolated from 2005 to 2016 were provided by the Linkou Chang Gung Memorial Hospital, Taiwan. Regardless of the illness diagnosed, we randomly picked clinical samples from epidemics in this time span. To prevent contamination, amplified viral stocks from human rhabdomyosarcoma cells were used for full-genome sequencing. Viral genomes were recovered using TRIzol LS reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The 59 samples collected before 2014 were sequenced by Sanger sequencing. Oligo-(dT)₂₀ was used to prepare poly(A)-containing viral cDNA using a ReverTra Ace -α- kit (Toyobo, Osaka, Japan). Overlapping amplicons covering the entire viral genome were amplified by different sets of primers^{18 (link),31 (link)}, and genome assembly was carried out using SeqMan software (DNASTAR, Inc., Madison, WI, USA). The five samples collected after 2014 were sequenced using the Illumina HiSeq platform for next-generation sequencing (NGS). NGS data preprocessing included the removal of low-quality and host reads. Using the Taiwanese B5 and C4 strains as an initial template, the viral genomes were assembled by an iterative mapping approach^{32 (link)}. A total of 64 genomes obtained in this study were deposited in GenBank with accession numbers MG756691–MG756754.