title: 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.
abstract: OBJECTIVE
  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.
  
  
  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 HPV-type 16. E6 full-length gene expression. In total there were 25 patients (40%) with expression of the HPV 16 E6*I gene and 30 patients with expression of the E6*II gene. The nested PCR method using S1/S2 primers detected 54 patients with the E6*I & E6*II transcripts in comparison to classical PCR which detected only 31 such transcripts.
  
  
  CONCLUSIONS
  Nested RT-PCR is the method of choice to determine the role of different E6/E7 splice products in HPV-associated carcinogenesis.
authors.affiliation: Gynaecology-Oncology Unit, Nelson R Mandela School of Medicine, Durban, South Africa.
authors.forename: M
authors.initials: M
authors.lastname: Moodley
authors.forename: J
authors.initials: J
authors.lastname: Moodley
authors.forename: C S
authors.initials: CS
authors.lastname: Herrington
authors.forename: S
authors.initials: S
authors.lastname: Sewart
authors.forename: R
authors.initials: R
authors.lastname: Chetty
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000032:analysis*; D027383:Papillomaviridae / Q000302:isolation & purification*; D011237:Predictive Value of Tests; D012097:Repressor Proteins / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D002583:Uterine Cervical Neoplasms / Q000821:virology*
chemical_list: D004279:DNA, Viral; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins