title: RNA extraction method is crucial for human papillomavirus E6/E7 oncogenes detection.
abstract: Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease. Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients' lesions and progression.
  
  
  
  This study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method.
  
  
  
  E6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%).
  
  
  
  Extraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.
authors.affiliation: Department of Immunology, Microbiology and Parasitology, Medicine and Odontology Faculty, University of Basque Country (UPV/EHU), Sarriena auzoa, 48940, Leioa-Bizkaia, Spain.
authors.forename: Nerea
authors.initials: N
authors.lastname: Fontecha
authors.affiliation: Clinical Microbiology and Infection Control Department, Basurto University Hospital, Bilbao, 48013, Spain.
authors.forename: Maria Carmen
authors.initials: MC
authors.lastname: Nieto
authors.affiliation: Department of Obstetrics and Gynaecology, Basurto University Hospital, Bilbao, 48013, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Andía
authors.affiliation: Department of Immunology, Microbiology and Parasitology, Medicine and Odontology Faculty, University of Basque Country (UPV/EHU), Sarriena auzoa, 48940, Leioa-Bizkaia, Spain.|Clinical Microbiology and Infection Control Department, Basurto University Hospital, Bilbao, 48013, Spain.
authors.forename: Ramón
authors.initials: R
authors.lastname: Cisterna
authors.affiliation: Department of Immunology, Microbiology and Parasitology, Medicine and Odontology Faculty, University of Basque Country (UPV/EHU), Sarriena auzoa, 48940, Leioa-Bizkaia, Spain. miren.basaras@ehu.eus.
authors.forename: Miren
authors.initials: M
authors.lastname: Basaras
mesh_terms: D000328:Adult; D055088:Early Detection of Cancer / Q000379:methods; D005260:Female; D006801:Humans; D008875:Middle Aged; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D030361:Papillomavirus Infections / Q000150:complications* / Q000473:pathology; D012367:RNA, Viral / Q000032:analysis* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000473:pathology / Q000821:virology; D055815:Young Adult
chemical_list: D015513:Oncogene Proteins; D012367:RNA, Viral
keywords: Cervical cancer biomarker; E6/E7 mRNA; High-risk HPV; Oncogenes; RNA extraction methods