Total rna purification system

Total RNA was isolated using the Total RNA Purification System (Invitrogen) following the manufacturer’s protocol. qPCR was performed with SYBR Green dye using an ABI Step One Real-Time PCR instrument (Applied Biosystems, Cheshire, U.K.). For relative quantification of gene expression, we used the ΔΔCt method. Results were normalized to expression of the control gene 36B4. Primers were designed according to published complementary DNA or genomic sequences. The primer sequences are shown in S1 Table.

Total RNAs isolated from EV-A71 infected RD cells treated with CW-33 alone or combined with IFN-β, using total RNA purification system (Invitrogen), were reverse-transcribed with oligo dT primer and SuperScript III reverse transcriptase kit (Invitrogen). To analyze gene expression in response to CW-33 alone or combined with IFNβ, quantitative PCR used cDNAs, primer pairs, and SYBR Green I PCR Master Mix. Pairs were forward 5′-GATGTGCTGCCTGCCTTT-3′ and reverse primer 5′-TTGGGGGTTAGGTTTATAGCTG-3′ for human 2′,5′-OAS (2′,5′-oligoadenylate synthetase), forward 5′-CATGGGCTGGGACCTGA CGGTGAAG-3′ and reverse primer 5′-CTGCTGCGGCCCTTGTTATT-3′ for IFNAR1 (interferon-α/β receptor 1), or forward 5′-AGCCACATCGCTCAGACAC-3′ and reverse primer 5′-GCCCCAATACGACCAAATCC-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Real-time PCR was completed by amplification protocol consisting of 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, 45 cycles at 95 °C for 15 s, and 60 °C for 1 min. Products were detected in ABI PRISM 7700 sequence detection system (PE Applied Biosystems); relative change in mRNA levels of indicated genes were normalized by mRNA level of housekeeping gene GAPDH.

Total RNA was isolated from frozen/fresh mouse liver tissues and primary hepatocytes using the Total RNA Purification System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Reverse transcription was carried out with 1 μg RNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Then, qPCR was performed using SYBR GreenER qPCR SuperMix (Invitrogen) according to the manufacturer’s instructions. mRNA expression levels were normalized to mouse β-actin as the internal standard. The primer pairs for the specific target genes were designed as listed in Supplementary Table S2.

For transcriptome analyses, mycelia were harvested by conidium cultivation in potato dextrose liquid medium at 25°C in darkness for 12 h. A pot culture experiment was used to harvest the infection samples. Sterile millet was inoculated with F. pseudograminearum mycelia, incubated at 25°C for 7 days, and then mixed with sterile soil (0.5% inoculation millet), in which wheat was subsequently grown. For negative controls, sterile millet was used. After 5 and 15 days, wheat roots from each pot were collected and washed thoroughly under running tap water and distilled water so that no soil particles remained. Samples of 6 μg of total RNA were extracted from each of the above-mentioned samples using the total RNA purification system (Invitrogen, Carlsbad, CA, USA). The total transcriptome was sequenced by the Gene Denovo Company (Guangzhou, China). Transcriptome raw data have been submitted and are publicly available in the NCBI database (accession number SUB5545839) (37 (link)).

Total RNA was extracted from the liver using the Total RNA Purification System (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Messenger RNA was reverse-transcribed using AccuPower CycleScript RT PreMix (Bioneer, Daejeon, Korea). Quantitative real-time PCR was performed with SYBR Green dye using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Cheshire, U.K.). Expression of the respective genes was normalized to the 36B4 signal as an internal control, and relative gene expression was quantitated by the comparative Ct method (ΔΔCt). The primer sequences of target genes are listed in Supplementary Table 1.