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. 2010 Jul 21;5(7):e11672. doi: 10.1371/journal.pone.0011672

Figure 2. Delayed infection kinetics of the ORF4−Tet+ and ORF4.STOP mutants as determined by quantitative RT-PCR.

Figure 2

The expression of the mRNA of the MHV-68 immediate-early gene Rta (ORF50) was examined by quantitative RT-PCR. Cells were infected at a multiplicity of infection of 10 for 1h at 37°C. Then, the inoculum was removed and fresh medium with PAA was added. Total RNA was isolated from infected cells 18 hours after infection and reverse transcribed. The resulting cDNA was used as template for PCR amplification of L8 and MHV-68 ORF50, respectively. Data are presented as relative expression of ORF50 after normalization to the corresponding L8 levels using the delta-delta Ct method. 18 hours after infection, the expression of the ORF50 mRNA was significantly reduced (*, p = 0,002; **, p = 0,025; Student's t-test) in cells infected with the ORF4 mutants when compared with the parental or revertant virus. Data shown are means ± SD of two independent experiments, each determined in duplicates.