Quantifying Retroviral and Epigenetic Markers

LINE-1, human endogenous retrovirus (HERVK14C), and SVA were assessed using cDNA prepared from the patients’ blood samples by quantitative polymerase chain reaction (qPCR), as previously described^{26 (link),49 (link)}. To examine the expression of methylation enzymes, including IFNs, JAK family members, TFs, and ISGs, qPCR was performed using specific primer pairs (Table S9). In brief, qPCR was performed using a THUNDERBIRD Probe and SYBR qPCR Mix (Toyobo). The expression level relative to that of a housekeeping gene was used in the analyses. Real-time PCR was performed using an ABI 7300 PCR thermal cycler (Applied Biosystems) under the following conditions: 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. The data were analyzed using the ΔΔCt method.