Construction of suppression subtractive hybridization library of renal cell carcinoma / 中华泌尿外科杂志

ABSTRACT

Objective To construct a suppression subtractive hybridization (SSH) library of human renal cell carcinoma. Methods Poly A + RNA was isolated from human renal cell carcinoma (RCC) tissues and matched normal kidney tissues, respectively. Then double strand cDNA were synthesized and restricted by Rsa I.RCC cDNA were divided into two groups and ligated with either adaptor 1 or adaptor 2.After RCC cDNA hybridized with normal kidney cDNA twice and underwent nested PCR,the PCR products were cloned into pT Adv vector and transformed E.coli TOP10F′.Some positive clones randomly picked up were digested and sequenced. Results The SSH library contained 414 positive clones. Random analysis of 265 clones with enzyme restriction showed that 246 clones contained cDNA fragments which were distributed mainly between 300～900 bp. Among 40 arbitrary clones derived from the above 246 clones, No.170 clone is a previously unknown sequence and the other 39 clones were derived from 35 known genes. Conclusions The quality of the SSH library of human RCC is reliable and its construction would lay the foundation for further screening differentially expressed genes in RCC.