Gene expression profile by Real-time PCR VLP-mediated transcriptional activation was tested for eighty four genes involved in Toll-like receptor (TLR)-mediated signal transduction using RT 2 Profiler PCR array (SABiosciences). The 96 well array format included mediators of TLR signaling including adaptors and proteins that interact with the TLRs, and members of NFKB, JNKp38, NF/IL6 and IRF signaling pathways downstream of TLR signaling. Briefly, 293 cells grown overnight in 60 mm dishes were exposed to 10 mg of purified VLPs suspended in 1 ml of OPTI-MEM (Invitrogen). ''Mock particles'' (see Methods, VLP harvest and purification) resuspended similarly and exposed to 293 cells served as negative control. The inoculums were adsorbed on the cell monolayers for 3 hours at 37uC when additional 1.5 ml of OPTI-MEM was added and the dishes further incubated. Twenty fours post VLP exposure, total cell RNA was extracted according to the manufacturer's (SABiosciences) instructions. The integrity of the RNA was verified by agarose gel electrophoresis and the same concentration of total cell RNA from the VLP-stimulated and ''mock'' stimulated cells were used for gene expression profiling by Real-time PCR using Eppendorf Mastercycler unit. The array plate included positive and negative controls for quality assurance, and three sets of housekeeping genes for normalization for data analysis. The fold-change in gene expression in the VLP stimulated 293 cells relative to the ''mock'' stimulated 293 cells was calculated by the DDCt method according to the manufacturer's instructions.