Analysis of isolating differentially expressed genes through cDNA RDA combined with cDNA arrays

Objective To investigate the feasibility and limitation of isolating differentially expressed cDNA fragments from human osteoblast-like MG-63 cells induced by 17 beta-estradiol (E_2) through modified cDNA representational difference analysis(RDA) combined with cDNA arrays.Methods The internal quality control of RDA was performed by subtractive hybridization of “tester” and “driver” amplicon prepared by cDNA all from E_2 treated MG-63 cells.RT-PCR for GAPDH was performed to evaluate the subtractive efficiency of RDA.The differentially expressed cDNA fragments were confirmed by Southern blot and “shotgun” Northern blot. After confirmation,the cDNA fragments were cloned into the pGEM-T easy vector and the subtractive cDNA libraries prepared in E.coli JM109 cells.The cDNA libraries were grown on LB/Amp+/X-gal/IPTG plates and white colonies were picked up randomly and individually grown in LB/amp+ medium in 96-well plates.After PCR,colonies were individually blotted onto a Hybond N membrane.The prepared array membranes were hybridized with “tester” amplicon,“driver” amplicon and the fourth subtractive products to evaluate its usage for further study.Results After the third subtractive hybridization,no amplified products was found in PCR of the internal quality control of RDA.Through cDNA RDA,3 upregulated and 2 downregulated fragments were expressed in the fourth subtraction hybridization using cDNA from MG-63 cells induced with and without E_2 as “tester” amplicons,respectively.Southern blotting showed that strong hybridization signals were found in “tester” amplicon and the subtractive products,but not in 癲river” amplicon.Two hybridization signals from MG-63 cells treated with E_2 were stronger than those from MG-63 cells without E_2 in “Shotgun” Northern blotting.The two hybridization signals from the same clone showed high consistency on hybridized membranes.Through array dot blotting,we obtained 120 differentially expressed clones including 56 upregulated and 64 downregulated ones.Conclusions It is an effective and fast method to isolate differentially expressed genes from human MG-63 cells induced by E_2 through modified cDNA RDA combined with cDNA arrays.