Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma

Objective To construct a renal cell carcinoma ( RCC) cDNA subtractive libra ry using suppression subtractive hybridization. Methods Polyadenylated RNA [Poly (A)(+) RNA] was isolated from tissues of R CC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors 1 and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cl oned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some und erwent sequence analysis and Northern blot to identify RCC specifically exp ressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes. Results A human RCC subtractive library with high subtractive efficiency wa s successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmi ds in the clones contained 50-400 bp inserts. Sequence analysis was perform ed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. N orthern blot analysis showed that RCC18 cDNA was highly expressed in RCC, b ut no signal could be detected in normal kidney. Using SMART RACE technique , we obtained the full length of the novel gene RCC18. Conclusions The constructed cDNA subtractive library of human RCC is a high ly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The no vel specifically expressed genes provided an important clue for studying th e mechanisms of occurrence and development of RCC.