Combination of PCR subtraction and cDNA microarray for differential gene expression profiling

PCR subtraction hybridization has been used effectively to enrich and singl e out differentially expressed genes. However, identification of these gene s by means of cloning and sequencing individual cDNAs is a tedious and leng thy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent det ection method. mRNA from human prolactin (hPRL) or human prolactin antagoni st (hPRL-G129R) treated and non-treated breast cancer cells was isolated, a nd cDNAs were synthesized and used for the PCR subtraction to enrich the di fferentially expressed genes in the treated cells. The PCR-amplified and su btracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membran e and identified by chemiluminescence. The results suggest that the strateg y of combining all three methods will allow for a more efficient, nonradioa ctive way of identifying differentially expressed genes in target cells.