Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens

Abstract

Background: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens.

Results: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%.

Conclusion: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Figure 1

Comparison of Standard Curves from Repeated Assays by HPV Type. This figure is a graphical comparison of the standard curves from each of the HPV 16 and HPV 18 assays. There were four assays performed for HPV 16 and two for HPV 18. The type-specific curves were not statistically different from one another, and those for HPV 16 are graphically identical. (Note: ln(SQ) = natural log of starting quantity.)

Figure 2

Standard Curve from HPV 16 E7 Assay. This figure represents the standard curve of the HPV 16 PCR. Note, the standard dilutions appear as blue circles on the graph, and the unknown samples appear as red squares. The calculated regression line of the curve is given above the graph. In this case, Y = -2.882X + 34.568. Setting Y equal to the C_T for the unknown, solving for X will give the starting quantity for the unknown sample.