IMR Press / EJGO / Volume 25 / Issue 1 / pii/2004108

European Journal of Gynaecological Oncology (EJGO) is published by IMR Press from Volume 40 Issue 1 (2019). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with S.O.G.

Open Access Original Research

Use of the nested reverse transcription-polymerase chain reaction for the detection of human papillomavirus 16 E6 transcriptional activity in cervical cancer: A technical perspective

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1 Principal Specialist, Gynaecology-Oncology Unit, Nelson R Mandela School of Medicine, Durban (South Africa)
2 Prof/Dir., MRC-Pregnancy Hypertension Unit, Nelson R Mandela School of Medicine, Durban (South Africa)
3 Prof., Department of Anatomical Pathology, University of Liverpool, Liverpool (UK)
4 Laboratory Scientist Department of Biological and Life Sciences, University of Liverpool , Liverpool (UK)
5 Professor of Pathology, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto (Canada)
Eur. J. Gynaecol. Oncol. 2004, 25(1), 51–54;
Published: 10 February 2004
Abstract

Aim: The aim of this study was to evaluate HPV 16 E6 expression using nested RT-PCR in cervical tumour tissue and compare this technique with standard RT-PCR in a group of patients using injectable contraceptive steroids. Patients and Methods: Tumour DNA was analysed for the presence and type of HPV by polymerase chain reaction (PCR) from 120 cervical cancer samples. Ribonucleic acid (RNA) was extracted from cervical tissue samples and cell-lines. Reverse transcrip­tion was carried out on all samples using reverse transcriptase enzyme to form single-stranded cDNA. The GAPDH (glyceralde­hyde-3-phosphate dehydrogenase) housekeeping gene was used. Results: The majority of patients had squamous cell carcinoma. Of 120 cervical tissue samples, there were 111 samples with con­firmed HPV 16 infection. RNA was extracted in only 86 samples. Of these, 23 samples contained genomic DNA. Of the remaining 63 patients, there were 53 patients who had expression of HPY-type 16, E6 full-length gene expression. In total there were 25 patients (40%) with expression of the HPV 16 E6*1 gene and 30 patients with expression of the E6*11 gene. The nested PCR method using S 1/S2 primers detected 54 patients with the E6*1 & E6*II transcripts in comparison to classical PCR which detected only 31 such transcripts. Conclusion: Nested RT-PCR is the method of choice to determine the role of different E6/E7 splice products in HPV-associated carcinogenesis.

Keywords
Nested RT-PCR
HPV-related cervical carcinogenesis
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