FIG. 1.
Relative quantitation of RNA packaged into HCMV particles with real-time RT-PCR. (A) Real-time RT-PCR was completed with RNA isolated as described in Materials and Methods from fibroblasts and cell-free virus following 72 h of infection. cDNA was synthesized with random hexamers, and reactions were completed with primers to numerous viral and cellular genes with SYBR green detection. The results are presented as the ratio of the CT value obtained from the virion RNA sample to the CT value obtained from RNA isolated from infected cells. The ratios were normalized to the ratio for UL21.5 and are presented as the inverse value. An estimate of the packaging efficiency is shown when 10-fold more RNA is packaged as well as 0.1-fold less RNA relative to UL21.5. The data are presented as the standard deviation from two experiments. (B) Northern blot analysis was completed with an equal volume of virion RNA and 2 μg of total RNA from infected cells. Specific transcripts were detected with 32P-labeled strand-specific probes to UL21.5, UL83, and UL107. Additional low-molecular-weight bands observed in virion RNA samples are indicated by lines. The locations of marker RNAs are indicated to the left of the gel, with sizes in kilobases.