Quantitative Analysis of Gene Expression

Total RNAs from infected GM-CSF-BMDC were extracted using RNeasy Micro Kit (Qiagen) following manufacturer’s instructions. CLN were stabilized in RNAlater (Qiagen) immediately after sampling. Organs were homogenized in RLT buffer (Qiagen) and then extracted using RNeasy Micro Kit (Qiagen) following manufacturer’s instructions. cDNAs were obtained by using Quantitech Reverse Transcription Kit (Qiagen) following manufacturer’s instructions using 300 ng of RNA as a matrix. qPCR were conducted using a 7500 Fast-Real-time PCR (Applied Biosystem) with SYBER Green (Takara) following manufacturer’s instructions. HPRT was used as housekeeping gene to determine ΔCt. Fold change compared to base line expression in uninfected cells, or control mice was determined using 2^-ΔΔCt method where ΔΔCt = (Ct_target-Ct_HPRT)_infected-(Ct_target-Ct_HPRT)_non-infected as previously described (Papadopoulos et al., 2016 (link)). mRNAs whose expression level was twice as high compared to control were considered as significantly up-regulated. Primers used in this study are listed in Table 1.