Figure 4

Detection of NA vRNA, cRNA and mRNA by primer extension assays. Total RNA from infected cells were harvested at 8 and 24 hr postinfection. The reaction conditions were identical to previously described assays [31], except fluorescent vRNA-specific primer (5'-Cy3-TGGACTAGTGGGAGCATCAT-3') and cRNA/mRNA-specific primer (5'-Cy5-TCCAGTATGGTTTTGATTTCCG-3') were used in the assays. The fluorescent products were resolved in 10% denaturing polyacrylamide gels and the images were analyzed by an imaging analyzer (Typhoon 8600 variable mode imager, Amersham Biosciences). Signals for the vRNA, cRNA and mRNA are shown as indicated. cRNA and vRNA signals of the NSNA-U were consistently higher than those of the All-U in independent attempts (Trials 1 and 2).