Identification of a novel gene marker specific for epithelial cells by utilizing a 3'-directed cDNA library.
To achieve reliability of molecular diagnosis using reverse transcription-PCR (RT-PCR), we established a unique method to search for a novel gene marker specific for colonic epithelial cells. Of eight candidate genes selected from a 3'-directed cDNA library in colonic mucosa, two genes were expressed in normal mucosa and cancer of the colon but not in either normal lymph node or normal liver tissue. Known sequences of these genes were reported to be located in the 3' noncoding region, and an additional sequence just upstream to gs04094 (one of the candidate genes) was determined. According to the newly identified sequence, we designed a new set of primers so that we could distinguish the DNA fragment amplified in RT-PCR from that in genomic PCR. RT-PCR using these primers demonstrated that gs04094 was expressed in all of 10 primary colon cancers and 4 liver metastases from colon cancer but in none of 5 normal lymph nodes, 10 peripheral blood samples, and 2 normal liver tissues. Sensitivity of this method was so high as to detect gs04094 mRNA in 10(-6) microg of colon cancer RNA per 1 microg of normal lymph node RNA. Thus, our strategy to search for a novel gene marker using 3'-directed cDNA library proved to be highly efficient.
Ohnishi
T
T
Department of Surgery II, Biomedical Research Center, Osaka University Medical School, Suita, Japan.
Tomita
N
N
Miyoshi
Y
Y
Shinoki
N
N
Tanaka
T
T
Kawabata
Y
Y
Sekimoto
M
M
Monden
T
T
Okubo
K
K
Matsubara
K
K
Monden
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003106:Colon / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D017931:DNA Primers; D016208:Databases, Factual; D004847:Epithelial Cells / Q000737:chemistry*; D015723:Gene Library; D005819:Genetic Markers; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry; D008113:Liver Neoplasms / Q000556:secondary; D008832:Microchemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D005819:Genetic Markers