Figure 4. Relative expression of transfer vector RNAs within the transfected cells.

Real time PCR analysis of (A) cytoplasmic transfer vector RNAs and (B) cytoplasmic β-actin RNA expression expressed as logΔRn verses cycle number. ΔRn is the target gene-specific fluorescence signal (FAM for MMTV-specific and VIC for ß-actin-specific sequences) normalized to the signal for the internal passive control, ROX (Normalized Reporter or Rn) from which the baseline target fluorescence has been subtracted (ΔRn = Normalized Reporter (Rn) - baseline). (C) Relative cytoplasmic transfer vector RNA expression in 293T cells after normalization with β-actin and luciferase expression. MK, Mock, transfected cultures with packaging construct, JA10 + the VSV-G-Env expression plasmid, MD.G + luciferase-expression vector, pGL3 except the transfer vector. RQ, Relative Quantification in log₁₀ units. The primers for detecting the transfer vectors were designed within the U5 region of the MMTV LTR, a region common to all transfer vector RNAs. Each sample was tested in duplicates with MMTV- and β-actin-specific probes and primers as described in Materials and Methods.