Comparison and optimization of in situ hybridization procedures yielding rapid,sensitive mRNA detections |
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| Institution: | 1. Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, 1210 Eisenhower Pl., Ann Arbor, MI, USA 48108;2. Department of Periodontics, School of Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78240, USA;3. Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, 150 College Street, Fitzgerald Building, Toronto, ON M5S 3E2, Canada;4. Department of Orthopaedics, RAYO, Carlisle Center for Bone and Mineral Imaging, School of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX, USA;5. Department of Pediatric Dentistry, School and Hospital of Stomatology, Peking University, 22 South Avenue, Zhongguancun Haidian District, Beijing 100081, PR China;1. Department of Molecular and Cell Biology and Biochemistry, Basic Veterinary Science, Faculty of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan;2. Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan;3. Education and Research Center for Fermentation Studies, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan |
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| Abstract: | This paper describes methods that are commonly used for performing mRNA in situ hybridizations. Each stage of the procedure has been analyzed to identify the parameters that most significantly affect the final cell morphology and sensitivity of the system. We have identified key elements of the procedure as the fixation employed, the type of polynucleotide probe and label chosen, and the detection system used. By optimizing these critical components, we have developed a procedure for performing mRNA in situ hybridizations that takes 2–4 hours and has a sensitivity of 1–10 molecules of mRNA per cell. This system has been used to detect levels of oncogene expression in normal bone marrow and peripheral blood. It is possible to detect the expression of three oncogenes (c-myc, c-sis, and c-abl) simultaneously in a small population of cells from the peripheral blood of leukemic patients. |
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