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2 protocols using ifih1

1

Quantifying Immune Response Transcripts in Neutrophils

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Total RNA was isolated using the RNeasy Mini kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA quality and quantification were determined by TapeStation 4200 (Agilent, St. Clara, USA) following the manufacturer’s instructions. Neutrophil total RNA was isolated using RNeasy Mini kit following the manufacturer’s instructions (Qiagen), and complementary DNA (cDNA) was synthesized using M-MLV Reverse Transcriptase (Thermo Fisher Scientific). RT-PCR was performed in duplicates using the cDNA and Platinum Taq polymerase (Thermo Fisher Scientific), 200 nM dNTP (Promega, Southampton, UK), 50mM MgCl2 (Thermo Fisher Scientific), and TaqMan primer/probe sets (Thermo Fisher Scientific). Samples were matched to a standard curve generated by amplifying serially diluted products using the same PCR reaction and normalized to GAPDH (Hs00266705_g1) to obtain the relative expression value. Real-time assays were run on FX96 Cycler (Bio-Rad). The following are the primes used: STAT1 (Hs01013996_m1), STAT2 (Hs01013115_g1), STAT3 (Hs00374280_m1), IFIH1 (Hs00223420_m1), IFIT (Hs00356631_g1), ISG15 (Hs00192713_m1), MX1 (Hs00895608_m1), IRF1 (Hs00971965_m1), SOCS1 (Hs00705164_s1), USP18 (Hs00276441_m1), IFI44 (Hs00197427_m1), IFI16 (Hs00986757_m1), and OAS (Hs00242943_m1) (all from Thermo Fisher Scientific).
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2

Gene Expression Analysis of Antiviral Pathways

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Total RNA was extracted by Trizol (Life Technologies) reagent according to the manufacturer's protocol. The acquired RNAs were treated with RNase-free DNase. Next, these RNAs were reverse transcribed to cDNA with oligo (dT). Precise gene manifestation was computed using SuperReal PreMix SYBR Green (FP204-02, TIANGEN, China). β-actin was used as the internal control for normalization. Following primers (Thermo Fisher) were utilized: IFIH1 sense (TCACAAGTTGATGGTCCTCAAGT), IFIH1 antisense (CTGATGAGTTATTCTCCATGCCC); IRF3 sense (AGAGGCTCGTGATGGTCAAG), IRF3 antisense (AGGTCCACAGTATTCTCCAGG); IRF7 sense (CCCACGCTATACCATCTACCT), IRF7 antisense (GATGTCGTCATAGAGGCTGTTG); IFIT1 sense (TTGATGACGATGAAATGCCTGA), IFIT1 antisense (CAGGTCACCAGACTCCTCAC); IFNB sense (GCTTGGATTCCTACAAAGAAGCA), IFNB antisense (ATAGATGGTCAATGCGGCGTC); M1 NS3 sense (GGGGAGGGCTTTCTTTGTCA), M1 NS 3 antisense (CACCCTGTCTTGTCTTTGCTG); β-actin sense (GATCATTGCTCCTCCTGAGC); β-actin antisense (ACTCCTGCTTGCTGATCCAC).
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