Rayscript cdna synthesis kit

After
a 24 h incubation, TRIzol reagent (Invitrogen) was used to extract
mRNA according to the instructions.^{45 (link),46 (link)} The RNA was
reverse-transcribed into cDNA using the Rayscript cDNA Synthesis KIT
(GENEray, GK8030, Shanghai, China) and subsequently employed in RT-PCR
reactions with SYBR Green (GENEray, GK8030, Shanghai, China). Table 4 describes the primers
of cytokines. Reactions were carried out on an ABI7500 apparatus (Applied
Biosystems Inc., USA). Before being compared with the control, the
level of mRNA to be tested was standardized to the level of GAPDH.
Relative game expression levels were quantitated using the ΔΔCt
method.^{48 (link)}

The A549 cells were seeded in a 6-well plate at 37°C with 5% CO₂, and then infected with influenza virus (PR8, 0.1 MOI). Following incubation for 2 h, the cells were treated with eleutheroside B1 (100 µg/ml). At 24 h post-infection, the cells were collected for the mRNA expression testing of selected genes [nuclear paraspeckle assembly transcript 1 (NEAT1) and L antigen family member 3 (LAGE3)] by RT-qPCR. The primer sequences of NEAT1 and LAGE3 are as presented in Table I. RNA was extracted with RnaExTM Total RNA Isolation Solution (GENEray, Inc.). The production of cDNA was then achieved using the Rayscript cDNA Synthesis kit (GENEray, Inc.) with 60 min at 37°C, and 5 min at 85°C. Subsequently, cDNA was used for qPCR using SYBR-Green Power qPCR PreMix (GENEray, Inc.). Primers of NEAT1 and LAGE3 were designed with Entrez Gene: 283131 and Entrez Gene: 8270. The thermocycling conditions were 1 cycle conditions including 10 min of initial denaturation at 95°C and 40 cycles of 10 sec denaturation at 95°C, 34 sec annealing at 60°C, 15 sec denaturation at 95°C, and 1 solubility curve cycle of 60 sec of annealing at 60°C, 30 sec annealing at 95°C, 15 sec annealing at 60°C. The method of quantification used was that of Livak and Schmittgen (2^−ΔΔCq) (39 (link)).

Ribosomal RNA depletion was performed using a 5′-phosphate-dependent exonuclease (GeneRead rRNA Depletion kit (Qiagen, Germany), which degraded transcripts with a 5′ monophosphate. Linear RNA depletion was performed using Ribonuclease R. The dosage ratio of Ribonuclease R to RNA is 3U:1 µg and the digestive conditions were 37°C for 30 min. According to experimental protocols, Rayscript cDNA Synthesis kit (GENEray, GK8030) was used for the reverse transcription. RNA fragments were used for sequential first-strand and second-strand cDNA synthesis, and the cDNA templates were enriched. Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep kit (Illumina, Inc., San Diego, CA, USA). Library quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) prior to sequencing. Total concentrations were determined using a Qubit 12.0 Fluorometer (Thermo Fisher Scientific, Inc.). Libraries were sequenced on the Illumina NextSeq 500 platform (Illumina, Inc.) with 2×150 bp paired-end reads. The libraries were constructed and sequenced by Shanghai Personal Biotechnology Co., Ltd. (www.personalbio.cn/; Shanghai, China).