Background: ThinPrep is a fluid-based technique for collection and processing of cytologic specimens. The present study was designed to determine whether the collection solution preserved RNA for molecular analysis. Methods and Results: Cervical cancer cell lines and cord blood lymphocytes were used to test the efficacy of various protocols for fixation, storage, and extraction of RNA. Total RNA was extracted and analyzed by denaturing gel electrophoresis. Preserved cells stored for 24 hours at room temperature or 4 degreesC had intact 28S and 18S ribosomal RNA. Both cellular and viral messenger RNAs were amplified from preserved samples by reverse transcription polymerase chain reaction (RT-PCR). Viral messenger RNA (mRNA) could be detected in a mixture of preserved cells containing 10% human papillomavirus (HPV) positive cells. RNA preservation in clinical samples was adequate for RT-PCR of cellular mRNA. Conclusions: Both experimental samples and clinical samples collected in the preservation media had intact total RNA. Amplification of both cellular and HPV ad mRNA was sucessful.
Background: ThinPrep is a fluid-based technique for collection and processing of cytologic specimens. The present study was designed to determine whether the collection solution preserved RNA for molecular analysis. Methods and Results: Cervical cancer cell lines and cord blood lymphocytes were used to test the efficacy of various protocols for fixation, storage, and extraction of RNA. Total RNA was extracted and analyzed by denaturing gel electrophoresis. Preserved cells stored for 24 hours at room temperature or 4 degreesC had intact 28S and 18S ribosomal RNA. Both cellular and viral messenger RNAs were amplified from preserved samples by reverse transcription polymerase chain reaction (RT-PCR). Viral messenger RNA (mRNA) could be detected in a mixture of preserved cells containing 10% human papillomavirus (HPV) positive cells. RNA preservation in clinical samples was adequate for RT-PCR of cellular mRNA. Conclusions: Both experimental samples and clinical samples collected in the preservation media had intact total RNA. Amplification of both cellular and HPV ad mRNA was sucessful.
Authors: Kristy A Warner; Erin L Crawford; Aiman Zaher; Robert J Coombs; Haitham Elsamaloty; Stacie L Roshong-Denk; Imran Sharief; Guillermo V Amurao; Yongsook Yoon; Amro Y Al-Astal; Ragheb A Assaly; Dawn-Alita R Hernandez; Timothy G Graves; Charles R Knight; Michael W Harr; Todd B Sheridan; Jeffrey P DeMuth; Robert J Zahorchak; Jeffrey R Hammersley; Dan E Olson; Samuel J Durham; James C Willey Journal: J Mol Diagn Date: 2003-08 Impact factor: 5.568