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Laboratory of Cancer Genetics [D. B. K., P. S. M., J. M. T.], National Center for Human Genome Research, Laboratory of Pathology [R. F. C., P. H. D., L. A. L., M. R. E-B.], and Surgical Branch [W. M. L.], National Cancer Institute, Bethesda, Maryland 20892
We report the construction of a plasmid-based cDNA library made from microdissected cells derived from prostatic intraepithelial neoplasia. Total RNA was extracted and converted to blunt-ended, double-stranded cDNA by oligo(dT)-mediated reverse transcription followed by linker addition. A linker-specific primer with UDG-compatible ends was used to amplify the cDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the clones derived from either known genes, anonymous expressed sequence tags, or novel transcripts with very little redundancy of screened clones. These results demonstrate the feasibility of constructing complex representative cDNA libraries from specific microdissected cell populations that represent microscopic precursor stages of cancer progression. This method should facilitate identification of transcripts specifically expressed in cells of a distinct histological origin and tumorigenic stage.
1 To whom requests for reprints should be addressed, at NCHGR/NIH Building 49, Room 4A15, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402-2015; Fax: (301) 402-3241.
Received 8/ 9/96. Accepted 10/17/96.
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