Construction of a differentially expressed genes subtracted cDNA library of human primary hepatocellular carcinoma by suppression subtractive hybridization

Objective To construct a differentially expressed genes subtracted cDNA library of human primary hepatocellular carcinoma (HCC) and paracancerous liver tissues. Methods Recently establishede new technique the suppression subtractive hybridization (SSH), to isolate the cDNA fragments of differentially expressed genes in HCC tissue and paracancerous liver tissues. Then these cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library, amplification of the library was carried out with transfomation of E.coli by high votage electroperforation. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. Results The amplified library contains more than 3000 positive bacteria clones. Random analysis of 100 clones with colony PCR method showed that 95% clones contained 100～600 bp inserts, these inserts might be the cDNA fragments of differentially expressed genes in HCC tissue. Conclusion A differentially expressed genes subtracted cDNA library of human HCC and paracancerous liver tissues is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific oncogenes or tumor suppressor genes of HCC.