Abstract
BackgroundFew 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.ResultsThe 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%.ConclusionThe 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.
Highlights
Few reports of the utilization of an accurate, cost-effective means for measuring human papillomavirus (HPV) oncogene transcripts have been published
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
Absolute quantitation of mRNA Eighteen cytobrush specimens were analyzed by real-time PCR for the presence of either HPV16 or HPV18 E7 mRNA (Table 1)
Summary
Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. 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. Infection with certain types of human papillomavirus (HPV), while a necessary cause of cervical cancer, does not guarantee an elevated risk of malignancy. The E7 oncogene exerts its transforming function by interrupting cell differentiation and inducing DNA synthesis [7]. It accomplishes this by interacting with the cellular tumor suppressor gene product pRB [8,9]. E7 can induce abnormal centrosome duplication [10,11] and chromatin condensation [12] possibly leading to chromosome instability
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