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The IFNA1 is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed for research and scientific applications, but a detailed description of its core function is not available at this time while maintaining an unbiased and factual approach.

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5 protocols using ifna1

1

Quantitative RNA Expression Analysis

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Total RNA content from 1 × 105 cells were extracted using PureLink® RNA Mini Kit (Applied Biosystems) per manufacturer’s protocol and cDNA conversion was done using High-Capacity cDNA RT Kit (ThermoFisher). Quantitative PCR reactions and analysis were performed using ViiA 7 (ThermoFisher). For BM APC experiments, cells were enriched using CD45 magnetic beads. TaqMan®FAM™ dye-labeled probes including GAPDH (Hs02786624_g1, Mm99999915_g1), GADD45A (Hs00169255_m1, Mm00432802_m1), DDIT3 (Hs00358796_g1, Mm01135937_g1), HERPUD1 (Hs01124269_m1, Mm00445600_m1), IFNA1 (Mm03030145_gH), and IFNB1 (Mm00439552_s1) (ThermoFisher). GAPDH was used to normalize signal expression. Fold change comparison were performed between control and treated samples using the ΔΔCT method as described31 (link).
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2

qPCR Analysis of Immune Genes

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qPCR was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems, Thermo Fisher Scientific, 4369016). Data were analyzed by the 2-DDCt method using the mean adult values as the reference. Primers/probes were from Thermo Fisher Scientific: MX1 (Hs00895608_m1), MX2 (Hs01550811_m1), IFNA1 (Hs00256882_s1), IFI44 (Hs00951349_m1), IFIT1 (Hs03027069_s1), IL17A (Hs00174383_m1), IFNG (Hs00989291_m1), RPLPO (4326314E).
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3

Quantitative Analysis of IFNA1 mRNA Expression

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Total RNA was isolated from 5 × 105 cells using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). Total RNA (1 μg) was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) to exclude amplification of genomic DNA then reverse transcribed into cDNA using the High Capacity cDNA RT Kit of Applied Biosystems (Carlsbad, CA, USA). The cDNA products were diluted at a ratio of 1:2, then amplified using Dream Taq DNA Polymerase (Thermo Fisher Scientific) and IFNA1 (Assay ID Hs. PT.49a.3184790.g, Integrated DNA Technologies, Coralville, IA, USA) gene-specific assay according to the manufacturer's instructions. Quantitative PCR was performed using the ABI StepOne Real-Time PCR System (Applied Biosystems) and cycle threshold values were determined using the StepOne v2.1 Software (Applied Biosystems). The relative amount of mRNA (2−ΔCt) was obtained by normalizing to the cyclophilin house keeping gene in each experiment.
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4

Quantitative PCR analysis of human liver

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Whole liver RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) including DNAse treatment according to manufacturer’s protocol starting with homogenization of liver tissue in RLT buffer. cDNA was generated by using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. Human specific gene expression was measured using Taqman primer/probe quantitative PCR, in TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Primer/probe combinations were purchased from Thermo Fisher Scientific; CXCL10 (Hs01124251_g1), CXCL9 (Hs00171065_m1), DDX58 (Hs01061436_m1), GAPDH (Hs00266705_g1), IFIT1 (Hs01911452_s1), ISG15 (Hs01921425_s1), IFNA1 (Hs00855471_g1), IFNA4 (Hs01681284_sh), IFNB1 (Hs01077958_s1), MX1 (Hs00895608_m1), OAS1 (Hs00973637_m1), RSAD2 (Hs00369813_m1), STAT1 (Hs01013996_m1), TLR3 (Hs01551078_m1). Expression of target genes was normalized to the expression of GAPDH using the formula 2−ΔCt, ΔCt = Cttarget−CtGADPH. cDNA from non-chimeric mouse livers was used as control to test cross-reactivity of housekeeping and target genes. Due to the difference in hepatocyte donor baseline expression levels of examined genes (Suppl. Fig. 1), fold changes of transcripts were calculated to those of non-infected humanized livers from mice transplanted with the identical hepatocyte donor.
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5

Quantitative Gene Expression Analysis

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Total RNA was isolated from transfected or infected cells using the Trizol RNA isolation method. cDNA was prepared using 1000ng of total RNA and random hexamer primers. Samples were incubated at 16°C for 30 minutes, 42°C for 30 minutes and 85°C for 5 minutes. Real-time PCR (Taqman) was used to analyze cDNA levels in transfected or infected samples. An ABI StepOnePlus Real Time PCR machine was used with the following program for 40 cycles: 95°C for 15 sec and 60°C for one minute. Relative expression was determined using the ΔΔCt method using 18S as the standard control. JunB, SERPINE, SMAD3, IFNA1, IFNB1, RSAD2, IRF7 and 18S primer/probe sets were obtained from Thermo Fisher Scientific.
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