Wenhua Zhou1, Katherine Geiersbach2, Barbara Chadwick3. 1. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah. 2. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. 3. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah. Electronic address: Barbara.chadwick@utah.edu.
Abstract
INTRODUCTION: Because of consistent demand for identification of molecular targets for diagnosis and treatment, ancillary tests are increasingly being developed for cytology specimens. One time-consuming step in this procedure is glass coverslip removal by xylene. Here we describe a method for rapid coverslip removal using liquid nitrogen. MATERIALS AND METHODS: Direct smears were prepared from residual pleural fluid, Diff-Quik stained, then covered with a glass coverslip. Coverslips were then removed by either immersing the slides in xylene for 3 to 4 days, or by a razor blade after rapidly freezing the slides in liquid nitrogen. The DNA or RNA was extracted, and the quantities of DNA and RNA were evaluated. The amplifiability of the DNA and RNA was also evaluated by real-time polymerase chain reaction (PCR) and real time reverse transcription (RT)-PCR. RESULTS: The concentrations of DNA isolated using the two procedures are not significantly different (2.79 μg/mL with liquid nitrogen method versus 2.56 μg/mL with xylene method, n = 5). Similar results were observed for RNA concentration (13.28 μg/mL versus 12.22 μg/mL). The DNA extracted from the two procedures can be amplified without significant difference. Similar results were obtained from real time RT-PCR for RNA samples. CONCLUSIONS: By removing the coverslip from the cytology slides using the liquid nitrogen method, DNA and RNA can be extracted from the slides without affecting the quantity and quality of the nucleic acids, and is equivalent to the xylene coverslip removal method.
INTRODUCTION: Because of consistent demand for identification of molecular targets for diagnosis and treatment, ancillary tests are increasingly being developed for cytology specimens. One time-consuming step in this procedure is glass coverslip removal by xylene. Here we describe a method for rapid coverslip removal using liquid nitrogen. MATERIALS AND METHODS: Direct smears were prepared from residual pleural fluid, Diff-Quik stained, then covered with a glass coverslip. Coverslips were then removed by either immersing the slides in xylene for 3 to 4 days, or by a razor blade after rapidly freezing the slides in liquid nitrogen. The DNA or RNA was extracted, and the quantities of DNA and RNA were evaluated. The amplifiability of the DNA and RNA was also evaluated by real-time polymerase chain reaction (PCR) and real time reverse transcription (RT)-PCR. RESULTS: The concentrations of DNA isolated using the two procedures are not significantly different (2.79 μg/mL with liquid nitrogen method versus 2.56 μg/mL with xylene method, n = 5). Similar results were observed for RNA concentration (13.28 μg/mL versus 12.22 μg/mL). The DNA extracted from the two procedures can be amplified without significant difference. Similar results were obtained from real time RT-PCR for RNA samples. CONCLUSIONS: By removing the coverslip from the cytology slides using the liquid nitrogen method, DNA and RNA can be extracted from the slides without affecting the quantity and quality of the nucleic acids, and is equivalent to the xylene coverslip removal method.