Construction of a differentially expressed genes subtracted cDNA library of eosinophils that came from pretreatment and post-treatment asthma by suppression subtractive hybridization

Objective To construct a differentially expressed genes subtracted cDNA library of eosinophils that came from asthma. Methods By established recently a new technique of suppression subtractive hybridization (SSH), to isolate the cDNA fragments of different expressed genes in eosinophils of pretreatment and post-treatment asthma. The cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library, amplification of the library was carried out with transformation of E. coli by calcium chloride than screened the transformants by blue and white clones. One hundred positive bacteria clones were randomly picked and identified by colony PCR. Results The amplified library contains more than 300 positive bacteria clones. Random analysis of 100 white clone by colong PCR method showed that 90% clones contained 100～500 bp inserts, these inserts might be the cDNA fragments of differentially expressed genes in eosinophils of pretreatment asthma. Conclusions A differentially expressed genes subtracted cDNA library of eosinophils that came from pretreatment and post-treatment asthma was constructed successfully by SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new specific differentially expressed genes of eosinophils in asthma.