Construction of suppression subtractive cDNA libraries of normal and cadmium-transformed cells

Objective To construct a subtractive cDNA library of differentially expressed genes in normal and cadmium-transformed cells.Methods The human bronchial epithelial cells(16HBE cells) transformed by cadmium chloride was used as a tester,and the normal 16HBE cells as a driver,to construct a cDNA library using suppression subtractive hybridization(SSH).The products were inserted into TA vector after two substractive hybridization and nested PCR.The clones were picked up randomly and analyzed with PCR.Results The total RNA and mRNA of purity and good integrity were extracted,and the duble-stranded cDNA was well produced.The link efficiency of cDNA and connector was greater than 25%.And finally the differentially expressed genes were enriched with substractive hybridization and nested PCR.After blue-white screening,the amplified library contained more than 1 200 white positive clones,and fifty of them were selected randomly and analyzed with PCR.Totally 96% of the clones had the inserted 100-600 bp segment and the segments might be the gene cDNA segments with differential gene expressions.Conclusion The subtractive cDNA library of differential genes in transformed 16HBE cells induced by cadmium was successfully established.