Reverse transcription system

EA.hy926 cells were treated as mentioned above (in Western blot analysis). Then, the total RNA of EA.hy926 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). To remove the genomic DNA, total RNA was treated with DNase I for 2 min at 42 °C following the manufacturer’s protocol. The RNA integrity was checked via electrophoresis on 1.0% agarose gels. The RNA purity was identified by the 260/280 nm optical density ratio, and RNA samples with an 260/280 nm optical density ratio greater than 1.9 were selected for later analysis. Next, cDNA was synthesized using a reverse transcription system (Vazyme, Beijing, China) [22 ].
qRT-PCR was performed using Biosystems 7500 Fast real-time PCR and SYBR Premix Ex Taq II (RR047, RR420, Takara, Tokyo, Japan) according to the manufacturer’s instructions. Specific primers were 5’-TCGGCACAAAAGCACTATATG -3′ (forward), 5’-ACAGGACAAGAGGACAAGGC-3′ (reverse) for ICAM-1 and 5’-GAAGGTCGGAGTCAACGGAT-3′ (forward) and 5′- CCTGGAAGATGGTGATGG GAT-3′ (reverse) for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers were designed with Primer Premier 5 software. The results are expressed as the relative ICAM-1 mRNA levels compared with the untreated control, which was considered 100%.

Total RNA was extracted using a TRIzol reagent (Invitrogen, 15596–026). First strain cDNA was synthesized using the Reverse Transcription System (Vazyme, R211-01). Real-time PCR detection for TGF-β1, TGF-β2, TGF-β3, and GAPDH was performed using an ABI PRISM 7500 Sequence Detection System (New York, USA) with the following program: 30 s at 95°C, followed by 40 cycles at 95°C for 5 s and annealing at 60°C for 34 s. Relative mRNA expression was calculated and normalized by GAPDH.

The total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was generated using a Reverse Transcription System (Vazyme, Nanjing, China) following the manufacturer’s instructions. A real-time quantitative PCR (qPCR) experiment was performed using the SYBR Green PCR Master Mix kit (Tiangen Biotech, Beijing, China) and the IQ5 light cycler system (Bio-Rad, Hercules, CA, USA). The parameters for qPCR have previously been described [41 (link)]. The wheat β-actin and AtActin2 genes were used as internal controls in wheat and Arabidopsis, respectively. The samples were assessed using three biological replicates. The relative gene expression was calculated using the comparative CT method [42 (link)].

TRIzol reagent (Biosharp, China) was used to extract total RNA from cell samples, and total RNA was then reverse transcribed into complementary DNA (cDNA) with the reverse transcription system from Vazyme (Nanjing, China). The quantitative Real-Time PCR was performed with SYBR Green (Vazyme, Nanjing, China) following the instructions, and the reaction system was as follows: cDNA: 1 μL, forward primer (10 μM): 0.5 μL, reverse primer (10 μM): 0.5 μL, qPCR Master Mix: 10 μL, RNase Free Water: 8 μL. The relative amount of each gene was normalized to the amount of GAPDH, and the relative expression of the target genes was calculated by 2^−ΔΔCT. To quantify the IAV RNA replication, IAV PB1 was probed with PB1 forward primer (F): 5’-ATGGAATATGACGCTGTTG-3’ and reverse primer (R): 5’-TTGGCTTGTGTTGAGAATAG-3.’ Sequence of primers specific for GAPDH was as follows: F: 5’-CTCTGGTAAAGTGGATATTGT-3’ and R:5’-GGTGGAATCATATTGGAACA-3.’ To quantify the relative mRNA level of M1 and M2, the used primers were listed: M1 F: 5’-TGGAGGTTGCTAATAAGA-3,’ R: 5’-CTGCAAATTTTCAAGAAGGTCATCTCT-3’; M2 F: 5’-GTCGAAACGCCTACCAGAAG-3,’ R: 5’-CTCTAGCTCTATGTTGACAAAATGACC-3.’

Total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen, Carlsbad, CA). The cDNA was synthesized in 20 µL of reaction mixture using Reverse Transcription System (Vazyme Biotech Co, Nanjing, China) at 50 °C for 15 min and 85 °C for 5 s. The quantity of mRNA was determined using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co, Nanjing, China). The primers used were—β-actin, Forward primer: 5′-GCGAGAAGATGACCCAGATC-3′, Reverse primer: 5′-GGATAGCACAGCCTGGATA-3′; TNF-α, Forward primer: 5′-GCCTCTGTGCCTTCTTTTGA-3′, Reverse primer: 5′-GCAACCTTTATTTCTCGCCA-3′; IL-1β, Forward primer: 5′-CGACACATGGGATAACGAGGCTT-3′, Reverse primer: 5′-TCTTTCAACACGCAGGACAGGTA-3′; and MCP-1, Forward primer: 5′-CAAACTGAAGCTCGCACTCTCGCC-3′, Reverse primer: 5′-ATTCTTGGGTTCTGGAGTGAGTGTTCA-3′. Amplification was implemented in 20 µL of reaction volume according to the manufacturer’s instructions. The relative mRNA levels were calculated by the $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\text{T}}}$ method.

Total RNA was extracted using RNA Isolater reagent (Vazyme Biotech, Nanjing, China) according to the manufacturer’s protocol. The remaining genomic DNA in RNA samples was removed by treating with genome DNA wiper mix (Vazyme Biotech, Nanjing, China) at 42 °C for 2 min. First-stranded cDNA was synthesized with 2 μg of purified total RNA using reverse transcription system (Vazyme Biotech, Nanjing, China). qRT-PCR was performed on a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) with AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). Reactions were run under following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 10 s, 60 °C for 15 s and 72 °C for 30 s. Rice 18 s-rRNA gene [76 (link)] was used as an internal control to normalize data and relative expression levels of genes of interest were calculated using the 2^-ΔΔCT method. Gene-specific primers used in qRT-PCR are listed in Additional file 4: Table S1.