Constructing The SSH CDNA Library In Colorectal Cancer Exfoliated Urothelial Cells

Objective: to construct colorectal cancer patient and normal human colorectal exfoliative cells in stool differentially suppression subtractive cDNA library.Methods: Collects the rectum and colon's cast-off cells from the colon cancer patient and in normal person's excrement sample, First gathers the colon cancer patient excrement sample which already diagnosed , And takes the healthy volunteer fresh excrement sample to take the comparison; Collection colon cancer patient and healthyvolunteer's cast-off cells. To find the differentially expressed genes in the colon cancer patient's cast-off cells compared with normal person's cast-off cells and make the differentially expressed cDNA subtracted library:(1) Total mRNA was isolated from cancer patient and normal human colorectal exfoliative cells in stool, respectively. Then double-strand cDNA were synthesized and restricted by HaeIII. cancer patientcolorectal exfoliative cells cDNA were divided into two groups and ligated with eitheradaptor 1 or adaptor 2. After hybridized with normal colorectal exfoliative cells cDNAtwice.(2) the PCR products were cloned into PGM-T vector and transformed to E.coliJM109.Results:Random analysis of 375 clones with enzyme restriction showed that330clones contained cDNA fragments which were mainly between 200~800bp. Theinserted rate reached to 88%. A subtracted cDNA library of differentially expressedgenes is successfully constructed with SSH.Conclusions: The quality of the SSH library of colorectal cancer patient colorectal exfoliative cells in stool is reliable and its construction would lay the foundation for further screening differentially expressed genes. Among 20 arbitrary clones derived from 13 known genes. 2 clones is a previously unknown sequence.