Isolation of RNA from residual BD SurePath liquid-based cytology specimens and detection of HPV E6/E7 mRNA using the PreTectt HPV-Proofer assay

• Published in: Journal of virological methods Vol. 154; no. 1-2; p. 220
• Main Authors: Dixon, Eric P, King, Lorraine M, Adams, Melissa D, Grønn, Petter, Murphy, Patricia G, Brown, Charlotte A, Brough, George H, Skomedal, Hanne, Malinowski, Douglas P, Fischer, Timothy J
• Format: Journal Article
• Language: English
• Published: Netherlands 01.12.2008
• Subjects: Female; Humans; Oncogene Proteins, Viral - genetics; Papillomaviridae - genetics; Papillomavirus Infections - diagnosis; RNA, Messenger - isolation & purification; RNA, Viral - isolation & purification; Specimen Handling - methods; Vaginal Smears
• ISSN: 0166-0934
• DOI: 10.1016/j.jviromet.2008.08.002

Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.