Title: Constructing the SSH cDNA library in urine exfoliated urothelial cells of bladder neoplasms and normal
Abstract: s Objective:To construct the SSH cDNA library from exfoliated urothelial cells of bladder neoplasms and normal.Methods:Total mRNA was extracted from BTCC and normal exfoliated urothelial cells in urine respectively.Then double-strand cDNA were synthesized and restricted by HaeⅢ.BTCC cDNA were divided into two groups and ligated with either adaptor 1 or adaptor.After hybridized with normal exfoliated urothelial cells cDNA twice and underwent nested PCR,the PCR products were cloned into PGM-T vector and transformed to E.coli JM109.Some positive clones randomly picked up were digested,sequenced and homologously analyzed.Results:The SSH library contained about 400 positive clones.Random analysis of 384 clones with enzyme restriction showed that 317clones contained cDNA fragments which were mainly between 200-900 bp.The inserted rate reached to 82.6%.A subtracted cDNA library of differentially expressed genes is successfully constructed with SSH.Among 20 arbitrary clones derived from the above 317 clones,No.243 clone is a previously unknown sequence and the other 17clones were derived from 19 known genes.Conclusions:The quality of the SSH library of BTCC is reliable and its construction would lay the foundation for further screening differentially expressed genes.
Publication Year: 2008
Publication Date: 2008-01-01
Language: en
Type: article
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