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

ObjectiveTo construct a renal cell carcinoma (RCC) cDNA subtractive library using suppres sion subtractive hybridization MethodsPolyadenylated RNA [Poly (A) RNA] was isolated from tissues of RCC and norm al 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 l and 2, and then h ybridized with normal kidney cDNA twice with two rounds of suppression PCR Sec ond round PCR products were cloned to T/A plasmid vectors to set up the subtract ive library One hundred clones were randomly picked to perform enzyme digest a nalysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes SMART RACE procedure was operated to clone full l ength novel RCC specifically expressed genes ResultsA human RCC subtractive library with high subtractive efficiency was successfull y set up The amplified library contains 350 positive clones Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones cont ained 50-400?bp inserts Sequence analysis was performed for 10 clones All t he 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies Northern blot analysis showed tha t RCC18 cDNA was highly expressed in RCC, but no signal could be detected in nor mal kidney Using SMART RACE technique, we obtained the full length of the nove l gene RCC18 ConclusionsThe constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and speci fic oncogenes or tumor suppressor genes of RCC The novel specifically expresse d genes provided an important clue for studying the mechanisms of occurrence and development of RCC