FastLane Cell SYBR Green Kit (200)

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Publication protocol

We previously reported differential protective activities of porcine type I IFNs against PRRSV and VSV [21]. Here we demonstrate that porcine type I IFNs have different levels of activity in suppressing the resurgence of PERV RNA in PK-15 cells. In brief, cells were cultured in flat-bottom 96-well plates to 95% confluence, and 10 µl of each IFN peptide solution (20 ng in culture medium or medium only for controls) was added to a well containing 90 µl of fresh medium. At 60 h after incubation, cell RNA was extracted and the resurgence of PERV RNA was examined with a FastLane Cell SYBR Green Kit (Qiagen, Valencia, CA). In brief, RNA from cells of cultured wells was prepared using the FastLane lysing buffer to stabilize cellular RNA and eliminate genomic DNA, and FastLane lysates were used directly as templates in SYBR Green-based real-time one-step RT-PCR to detect the resurgence of PERV RNA using the published primers specifically for the PERV-γ1 subtype (including PERV-A, -B and –C) [33]. Real-time RT-PCR data were processed as above and plotted to show fold changes compared with control cells.

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