Mx3000p machine

Total RNA from stably infected PANC-1cells was extracted using the RNA simple Total RNA Kit (DP419, TIANGEN Biotech, Beijing). The first-strand of the cDNA was synthesized from total RNA using Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed with the following specific primers of KLK7: sense, 5′-ACCTCATGCTCGTGAAGCTC-3′; anti-sense, 5′-CCGGAGACAGTACAGGTGGT-3′. For GAPDH analysis (inner control), the following primers were used: sense, 5′-CAAGGTCATCCATGACAACTTTG-3′; anti-sense, 5′-GTCCACCACCCTGTTGCTGTAG-3′. Real-time PCR was performed on an Agilent Statagene Mx3000P machine using the SYBR Green II Dye detection system (SYBR^® Premix Ex Taq™ II, RR820A). Relative levels of gene expression were normalized against the level of GAPDH.

Seven representative genes of DEGs in the RNA-seq data were selected for validation by quantitative reverse-transcriptase PCR (qRT-PCR). All gene specific primers used in this study were designed by Primer Premier 5.0 software and listed in Table 1. Total RNA was extracted from control and PM2.5 treated sample sets using TRIzol Reagent (Ambion, Life technology, USA). Double-stranded cDNA was synthesized from 1 μg total RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was performed in triplicates using SYBR® Premix DimerEraser (TaKaRa Biotechnology, Dalian, China) with a Stratagene Mx 3000P machine (Agilent, CA, USA). GAPDH was chosen as the housekeeping gene in this study. The mRNA fold changes were calculated using the 2^-△△Ct method comparing △Ct of PM2.5 treated cells to △Ct of control untreated samples. Ct values were calculated using MxPro Mx3000P software version 4.01 (Stratagene, Agilent) applying automatic baseline and threshold settings.

Viral RNA was extracted from 200 μL of serum sample using the PureLink Viral RNA Mini Kit (Life Technologies, USA) according to the instructions from the manufacturer and immediately subjected to real-time qRT‒PCR. Real-time qRT‒PCR was performed with a Brilliant II SYBR Green qRT‒PCR Low ROX Master Mix system (Agilent, USA). In brief, a 25 μL mixture containing 5 μL of sample RNA, 0.25 μM forward and reverse DENV detecting or molecular-typing primers each, 2 × Brilliant II SYBR Green qRT‒PCR Low ROX Master Mix, RT/RNase Block Enzyme Mixture and RNase-free water was assayed in an Mx3000P machine (Agilent, USA). Dengue-specific primers for DENV RNA detection were DN-F: CAA TAT GCT GAA ACG CGA GAG AAA and DN-R: CCC CAT CTA TTC AGA ATC CCT GCT. Serotype-specific primers for molecular serotyping were DN-F: CAA TAT GCT GAA ACG CGA GAG AAA, D1-R CGC TCC ATA CAT CTT GAA TGA G, D2-R: AAG ACA TTG ATG GCT TTT GA, D3-R: AAG ACG TAA ATA GCC CCC GAC and D4-R: AGG ACT CGC AAA AAC GTG ATG AAT. The criteria of a positive control were a threshold cycle (C_t) value ≤ 30 and a Tm ≥ 79 °C, while a negative control had a C_t value ≥ 40 and a Tm < 79 °C. For the samples, a Ct value of ≤ 30 or a Tm ≥ 79 °C was considered positive [17 (link)].

For the quantitative analysis of Egr1 mRNA levels, real-time PCR analysis was performed with the qPCRBIO SyGreen Mix (PCR Biosystems protocol, PCR Biosystems Ltd, London, UK) on a Mx3000P machine (Agilent, Santa Clara, CA, USA) using the 2^−ΔΔCT method for quantitation. For this analysis, reverse transcription reactions (see above) were conducted using a mixture of oligo (dT) and an 18S-specific primer in order that 18S RNA (template diluted 1/1000 for QPCR) could be used for reference^{58 (link)}. This approach for normalization was adopted due to the reported widespread effects of RBFOX2 across the transcriptome^{27 (link)}.

RNA was extracted from OS tissue as previously described [43 (link)]. RNA was also extracted from cell lines with the Isolate II RNA kit (Bioline). cDNA was synthesised with the Tetro kit (Bioline) with oligo-(dT) primers. Both SYBR-green and multiplex based probe qPCR were performed on the Stratagene Mx3000P machine (Agilent Technologies) with gene-specific primers (S3 Table). The relative gene expression was normalized to the HPRT housekeeping gene and calculated by the 2^-ΔCT method.