An Improved Method for Construction of Directionally Cloned eDNA Libraries from Microdissected Cells

Here, we developed an improved method for constructing microdis sected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10' unidirectional recombinants with an average insert size of 500 bp. Single pass sequencing of 100 clones with the 11 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, se quences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any eDNA library. These results dem onstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.