FIG. 6.

Quantitation through real-time RT-PCR of E1∧E4-containing transcript levels within undifferentiated NHK cells containing recombinant HPV-31 mutants. (A) The concentration of E1∧E4 transcripts was determined through real-time RT-PCR using 2.0 μg of total RNA isolated from NHK lines containing recombinant genomes. Total RNA was isolated from lines placed in semisolid medium for 2 and 24 h. Quantitation was completed through SYBR green I dye incorporation into the amplicon, followed by quantitation of fluorescence. The RNA standard was synthesized through in vitro transcription reactions from an E1∧E4,L1 cDNA and amplified using the same primer set. Levels of E1∧E4-containing transcripts were determined from a standard curve (Stds). Similar results were seen in multiple experiments. (B) Quantitation was completed using a primer set which spans E1∧E4 using a donor splice site at nt 877 and an acceptor at nt 3295.