Nested polymerase chain reaction (nPCR) and RNase protection assays (RPAs). A: Representative autoradiographs of liquid hybridizations run 2X following nested polymerase chain reaction (nPCR) assays for MCMV immediate-early gene 1 (Eco RI E) DNA in the viscera of E18 embryos from a C.B-17 SCID mouse injected with 103 PFU of MCMV at E4. Four embryos [lanes A-D] demonstrated MCMV amplification; the fifth fetus [lane E] was MCMV DNA negative. Negative control lanes contained AE elution buffer (see text). MCMV control lanes contained AE elution buffer and 250 ag-250 fg of MCMV DNA Eco RI E. Note that nPCRs exhibited saturation for the positive controls. B-C: Representative cytokine RPAs for the brains of E18 fetuses following maternal IP injection at E4 with uninfected salivary gland suspension (USGS) or 103 PFU of MCMV (MCMV). In each lane 10 μg of total brain mRNA from a single E18 fetus was hybridized with one of two probe sets designed to detect 10 proinflammatory cytokines and 6 corresponding cytokine receptor transcripts, as well as mL32 and GAPDH housekeeping controls (see text). Labeled probe sets were used as size markers in the right lane of each film.