Ifna1

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 = Ct^target−Ct^GADPH. 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.

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.