Profiling EBV Gene Expression in Tree Shrew PBMCs

Total RNA was extracted from tree shrew PBMCs with the RNAsimple Total RNA Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol and quantified using a NanoDrop2000 instrument. Then, 500 ng of RNA was reverse transcribed to cDNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).RT-PCR was performed with a PCR Mix(Invitrogen, USA) according to the manufacturer’s protocol. The expression of BZLF1,LMP1,EBNA1,EBNA2 and EA was determined by RT-PCR using the primers,including 4 primers we designed for RT-PCR, as follows:BZLF1: 5′-GCG GAC AAA AAT CAG GCG TT-3′ and 5′-GAA AAT GCC GGG CCA AGT TT-3′; LMP1:5′-AAT TTG CAC GGA CAG GCA TT-3′ and 5′-AAG GCC AAA AGC TGC CAG AT-3′; EBNA2: 5′-AAC TCC TGG CCC ATC CAA TG-3′ and 5′-GGA GGG GCG AGG TCT TTT AC-3′; EA: 5′-AAC GAG CAG ATG ATT GGG CA-3′ and 5′-CGT GGT GAC GTA GAG ATC CG-3′ and 1 primers previously reported [60 (link)]: EBNA1: 5′-GTC ATC ATC ATC CGG GTC TC-3′ and 5′-TTC GGG TTG GAA CCT CCT TG-3′.The “housekeeping gene” β-actin was amplified as an internal control using the primers previously described [61 (link)], and cDNA from B95–8 cells was used as a positive control.