Figure 1. Quantitative real-time PCR of HPV16 URR to determine HPV16 viral load in DNA isolated from cervical precancer and cancer tissue biopsies . Genomic DNA of HPV16 positive cervical precancer and cancer samples was amplified by type-specific HPV16 URR primers using Biorad iQ SYBR Green supermix (as described in Methods). Two-fold serial dilution of WHO HPV16 international standards starting from 5x104 copies/reaction in C33a DNA diluents were used as reference (upper panels). Amplification of p53 exon5 which was used as normalization control for genomic DNA input (lower panels). DNA amplification threshold cycle analysis (left panels); standard curve analysis for determining the efficiency of reaction and calculation of viral copy number and quantitation of host genome equivalents (middle panel); Melt curve analysis showing specificity of HPV16 and p53 exon 5 amplicons were performed in each run (right panel).