Suppression subtractive hybridization for cloning of renal cell carcinoma relative genes

Objective To construct a cDNA subtractive library of human renal cell carcinoma (RCC) with suppression subtractive hybridization technique.The library contains only the differently expressin cDNAs between RCC and normal kidney. Methods Poly (A)+RNA were isolated from RCC and normal kidney tissues.Single strand cDNAs and double strand cDNAs were synthesized in turn.After enzyme restriction,cDNAs 600bp were obtained.RCC cDNAs were then divided into two groups and li gated to the specific adaptor l and adaptor 2 respectively.After RCC cDNAs were hybridized with normal kidney cDNA twice and underwent two times of nested PCR and was then connected with arms of T/A plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain Top 10F'. Results Human RCC subtractive library with high subtractive efficiency was set up successfully.The amplified library contains 350 positive clones.Random analysis of 200 clones with enzyme restriction shows that 190 plasmids in the clones contain 50～400bp inserts.Sequence analysis were performed in 15 clones.All of the sequences are unknown before. Conclusions The 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.The novel genes cloned proved to be an important clue for researching the mechanism of occurrence and development of RCC.