T A Tarkowski1, M S Rajeevan, D R Lee, E R Unger. 1. Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
Abstract
BACKGROUND: Molecular diagnosis requires the ability to obtain high-quality nucleic acids that are representative of the disease state. We evaluated the recovery and detection of limiting amounts of viral oncogenic RNA from cells fixed in liquid-based cytology media. METHODS AND RESULTS: Serial dilutions of a human papillomavirus (HPV)-positive cell line fixed in a liquid media was used as a model system. Total nucleic acid (TNA) extraction produced RNA with clearly visible ribosomal bands even after one year of storage. These TNA extracts, treated with DNase-I, were used in an RT-PCR assay for HPV-16 E6-E7 oncogenic transcripts. With chemiluminscent Southern blot detection, samples with one HPV-positive cell in 30,000 were consistently detected. CONCLUSION: PreservCyt-fixed cells can yield RNA suitable for molecular assays even after one year of storage.
BACKGROUND: Molecular diagnosis requires the ability to obtain high-quality nucleic acids that are representative of the disease state. We evaluated the recovery and detection of limiting amounts of viral oncogenic RNA from cells fixed in liquid-based cytology media. METHODS AND RESULTS: Serial dilutions of a human papillomavirus (HPV)-positive cell line fixed in a liquid media was used as a model system. Total nucleic acid (TNA) extraction produced RNA with clearly visible ribosomal bands even after one year of storage. These TNA extracts, treated with DNase-I, were used in an RT-PCR assay for HPV-16 E6-E7 oncogenic transcripts. With chemiluminscent Southern blot detection, samples with one HPV-positive cell in 30,000 were consistently detected. CONCLUSION: PreservCyt-fixed cells can yield RNA suitable for molecular assays even after one year of storage.
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