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Characterization of RNA in cytologic samples preserved in a methanol based collection medium.
Dimulescu I, Unger ER, Lee DR, Reeves WC, Vernon SD.Characterization of RNA in cytologic samples preserved in a methanol based collection medium.
Molecular Diagnostics, vol. 3, pages 67-72, 1998.
Summary
This is the first paper published from CDC's Molecular Epidemiology program. To apply the tools of molecular biology to patient samples from large scale epidemiology studies, simple effective methods for collection and preservation of sample nucleic acids must be developed and tested. This paper established that the collection methods that we will use in CFS studies in fact adequately preserves mRNA.
Abstract
Background: ThinPrep is a fluid-based technique for collection and processing of cytologic specimens. The present study was designed to determine if the collection media 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 C had intact 28S and 18S ribosomal RNA. Both cellular and viral messenger RNAs were amplified from preserved samples by reverse transcription PCR (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 mRNA was successful.
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