Construction of a representative cDNA library from prostatic intraepithelial neoplasia.
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.
Krizman
D B
DB
Laboratory of Cancer Genetics, National Cancer Institute, Bethesda, Maryland 20892, USA.
Chuaqui
R F
RF
Meltzer
P S
PS
Trent
J M
JM
Duray
P H
PH
Linehan
W M
WM
Liotta
L A
LA
Emmert-Buck
M R
MR
D000230:Adenocarcinoma / Q000737:chemistry / Q000235:genetics*; D002278:Carcinoma in Situ / Q000235:genetics* / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D018450:Disease Progression; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger