Cloning of renal cell carcinoma relation gene: Construction of a cDNA subtractive library of human renal cell carcinoma and its significance

To construct a cDNA subtractive library of human renal cell carcinoma (RCC) with technique called suppression subtractive hybridization. The library only contains the differently expressing cDNAs between RCC and normal kidney. Poly (A)⁺ RNA were isolated from cell lines of RCC and normal kidney respectively. Moreover, single-strand cDNAs and double-strand cDNAs were synthesized in turn. After enzyme restriction, cDNAs between 400600 bp were obtained. RCC cDNAs then were divided into two groups and ligated to the specific adaptor 1 and adaptor 2 respectively . After RCC cDNAs hybridized with normal kidney cDNA twice and underwent two times of nested PCR, then with arms of T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with the E. coli strain Top 10F′. Human RCC subtrctive library with high subtractive efficiency was set up sucessfully. The amplified library contains 6 500 positive clones. Random analysis of 350 clones with enzyme restriction shows that all plasmids in the clones contain 400-600 bp inserts. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays solid foundation for screening and cloning new and specific oncogenes or tumor suppressor genes of RCC.