Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA |
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| Institution: | 1. Department of Epidemiology, Center for Public Health, Medical University of Vienna, Vienna, Austria;2. Department of Livestock and One Health, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK;3. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA;4. Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK;5. Channing Division of Network Medicine, Department of Medicine, Brigham and Women''s Hospital and Harvard Medical School, Boston, MA, USA;6. Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA;7. Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA;8. Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA;9. Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA |
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| Abstract: | The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes. |
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