Construction of suppressive subtractive cDNA library of human pancreatic cancer.

Objective To construct a suppressive subtractive cDNA library of differentially expressed genes of human pancreatic cancer and normal pancreas tissues by suppressive subtractive hybridization (SSH). Methods Total RNA and mRNA were isolated from human pancreatic cancer (as tester) and normal paracancerous pancreas tissues (as driver), respectively. Then double-strand cDNA were synthesized. After Rsa I digestion, the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2R respectively. After tester cDNA was hybridized with driver cDNA twice and underwent nested PCR twice, the cDNA fragments of differentially expressed genes of human pancreatic cancer were isolated. Then the fragments were cloned into T/A vector and transformed E. coli TOP 10F', screened through the blue-white screening system, and then positive clones were confirmed by PCR method to set up the suppressive subtractive cDNA library. Results The amplified library contained approximately 257 white bacteria clones. Random analysis of 50 white clones with PCR amplification showed that 47 clones contained inserted fragments. The positive rate was 94%. The size of inserted fragments was between 300 - 600 bp. Conclusions A suppressive subtractive cDNA library of human pancreatic cancer is constructed successfully. The library would lay the foundation for further screening and cloning differentially expressed genes in human pancreatic cancer.